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http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_3
Team:HokkaidoU Japan/Notebook/aggregation Week 3
2012-09-27T03:58:23Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===July 16th===<br />
<div class="hokkaidou-section"><br />
Ag43, dT<br />
====Electrophoresis====<br />
Results of digestion at 15th.<br />
<br />
[[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&amp;P-1.jpg|thumb|Digestion result image]]<br />
Product:Ag43(K346007)=3120bp and 2070bp<br />pT7-RBS on pSB1K3=2247bp<br />
<br /><br />
We confirmed there are some kinds of restriction enzyme site in K346007 (digested with SpeI, PstI), and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI.ãBalance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.<br />
<br />
====Gel extraction====<br />
Gel extraction for digestion products. Used FastGene Gel&PCR extraction kit(NipponGenetics) andãgot 50 ul of DNA solution.<br />
<br />
====Ethanol precipitation====<br />
Ethanol precipitation for digested and extracted products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW.<br />
<br />
<br />
dT(B0015) would be amplifiedãincorrectly and we couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.<br />
====PCR====<br />
PCR for dT(B0015)<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp_up forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(200bp_down Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
[[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]<br />
<br />
We migrated B0015 of plasmid extraction product, digestion product, and PCR product.<br />
dT done PCR would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.<br />
<br />
====Gel extraction====<br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
<br />
====Digestion====<br />
'''Digestion for dT which amplified with PCR.'''<br />
Digested with XbaI and PstI.<br />
<br /><br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ag43<br />
'''Digestion result of Ag43 was incorrect'''. We digested Ag43 once more time.<br />
====Digestion====<br />
Digestion for Ag43 with SpeI and PstI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43 DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120716 Ag43d- Ag43d+.jpg|thumb|Digestion result image]]<br />
<br />
There are same results with digestion result of recent. '''We thought PstI would cut different site'''.<br />
What is this 500bp fragment????<br />
<br />
====Gel extraction====<br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50ul of DNA solution.<br />
<br />
====Liquid Culture====<br />
Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.<br />
<br />
'''Colonies which have Ag43-dT on pSB1T3 were closely existed so we would picked up two or more colonies.''' <br />
#Picked up one (or tow?) colony from single colony isolated platesãby platinum loop.<br />
#Dipped into 2 ml of LBC and LBT.<br />
#Incuvated.<br />
<br />
<br style="line-height: 0; clear: both;" /><br />
<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===July 17th===<br />
<div class="hokkaidou-section"><br />
Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3<br />
<br />
====Plasmid extraction====<br />
Plasmid extraction for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products incuvated from yesterday(16th).<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
====Electrophoresis====<br />
Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And plasmid extraction products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated.<br />
<br />
If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp.<br />
And if ligation and transformation, plasmid extraction were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp. <br />
[[image:HokkaidoU2012 120717 Ag43-Ag43d+(E&amp;S)-(Ag43-dT on pSB1T3)-(pT7-RBS on pSB1C3).jpg|thumb|Digestion and plasmid extraction result image]]<br />
From this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. '''would PstI not work correctly?''' <br />
<br />
'''We planed to use dT as vector and Ag43 as Insert.''' A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.<br />
<br />
About plasmid extraction products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought '''which means we failed single colony isolation then resuspended two another E.coli colonies''' another ligated DNA were transformed. <br />
In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.<br />
<br />
To confirm plasmid extraction products were really ligated correct DNA fragments, first we extracted Ag43-dT on pSB1T3(low bp band) from TBE gel and digested with EcoRI and PstI.<br />
<br />
'''dT Vector plasmid change'''<br />
To use dT as a vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.<br />
<br />
====Digestion====<br />
Digestion to change the plasmid backbone.<br />
Used DNA solution as PCR product(did at 16th) and digestioned pSB1T3 were already exist.<br /><br />
<br />
Digestion mix<br /><br /><br />
<br />
double digestion(EcoRI and PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717-dt-digestion-print.jpg|thumb|Digestion results]]<br />
<br />
We couldn't confirm digestion results.<br />
<br />
pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3<br />
<br />
====Electrophoresis and gel extraction====<br />
[[image:HokkaidoU2012 120717-ag43-dt-psb1t3.jpg|thumb|plasmid extraction result]]<br />
'''Gel extraction for Ag43-dT on pSB1T3.'''<br /><br />
We cut low bp band (see image below).<br />
Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
<br />
====Digestion====<br />
<br />
Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not.<br />
<br /><br /><br />
<br />
'''Ag43-dT on pSB1T3(30 ng/ul)'''<br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
'''pT7-RBS on pSB1C3(40 ng/ul)'''<br />
<br /><br /><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717 Ag43 dT(d-,d+E,d+P.jpg|thumb|Digestion result]]<br />
<br />
From this digestion results, <br />
*Cut with EcoRI,PstI and E&P(EcoRI and PstI) showed different base pair bands compared with d-(digestion minus).<br />
*Cut with E&P showed two DNA bands: about 2000bp and 500~1000 bp.<br />
*All of digestion products existed lower place than correct band.Correct DNA would show about 5000bp(EcoRI and PstI), 3000bp and 2000bp (E&P). <br />
<br />
<br style="line-height: 0; clear: both;" /><br />
<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 18th===<br />
<div class="hokkaidou-section"><br />
'''We decided change plasmid backbone of dT pSB1AK3 to pSB1T3.'''<br /><br />
If we cut Ag43-dT on pSB1AK3 with EcoRI and PstI to confirm insert DNA, DNA fragment base pair resemble each other(about 3000bp), so then we can't identify them.<br />
<br />
====digestion====<br />
'''Digestion to confirm what kind of restriction enzyme sites Ag43(K346007) has.'''<br />
<br /><br /><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|- <br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120718-k346007-digestion print.jpg|thumb|Digestion result]]<br />
From this result, we doubted '''are there one or more another PstI resutriction enzyme site except BioBrick suffix'''?<br />
<br />
====PCR====<br />
PCR to confirm what DNA fragment is ligated to vector as insert.<br />
We used PCR for pT7-RBS on pSB1C3. So the result would be 80~90bp band.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Biobrick prefix forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(Biobrick suffix Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
[[image:HokkaidoU2012 120718 pT7-RBS on pSB1C3 EX-F PS-R PCR.jpg|thumb|PCR product]]<br />
<br />
====Liquid Culture====<br />
Liquid culture for single colony isolated(19hrs incubated) Ag43-dT on pSB1T3.<br />
#Prepared 2 ml LB.<br />
#Added Tet.<br />
#Resuspended one colony.<br />
#Incuvated 16 hrs.<br />
<br />
We decided to start another ligation plan. First we digestion Ag43(K346007) and dT(B0015) with EcoRI&SpeI and EcoRI&XbaI then ligate. Ligation product cut with XbaI&SpeI and HindiIII. HindiIII can cut the site which is exist in pSB1AK3 then we can confirm Ag43-dT(about 3000bp) and pSB1K3(3000bp: cutting fragment will be about 1000bp). Next we digest pT7-RBS on pSB1C3 with SpeI only and ligate X-Ag43-dT-S and S-pSB1C3-pT7-RBS-S. If that go well, we can get pT7-RBS-Ag43-dT on pSB1C3 plasmid DNA which has never been digested with PstI. <br />
<br />
====Liquid culture====<br />
Liquid culture for Ag43(K346007)<br />
<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===July 19th===<br />
<div class="hokkaidou-section"><br />
===Plasmid extraction===<br />
Plasmid extraction for Ag43-dT on pSB1T3 and Ag43(K346007) which is inserted in pSB1C3.<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
===Electrophoresis===<br />
Electrophoresis for Ag43-dT on pSB1T3[about 5400bp] and Ag43(K346007)[about 5200bp] plasmid extraction products.<br />
[[image:HokkaidoU2012 120719 Ag43-dT on pSB1T3 and Ag43(K346007) mini-prep product.jpg|thumb|plasmid extraction result]]<br />
From this plasmid extraction result, Ag43-dT on pSB1T3 showed obviously higher DNA band than we estimated. It means '''Ag43-dT and pSB1T3 were misligated.''' And Ag43(K346007), we thought '''Ag43(K346007) DNA band existed in correct area''' because this DNA has about 5200bp and plasmid DNA migrates more far than linear DNA. Additionally, one weak 10kbp band existed in Ag43(K346007) lane.<br />
<br />
===Digestion===<br />
We conducted two digestion.<br />
*Digestion for Ag43(K346007) cut with EcoRI and SpeI. This DNA is for confirmation of PstI restriction enzyme cutting site.<br />
*Digestion for Ag43(K346007) and dT(B0015) cut with EcoRI & SpeI and EcoRI & XbaI.<br />
But we cut dT PCR product which didn't have plasmid vector so we retry digestion of dT after.<br />
<br /><br /><br />
Insert Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|16 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Vector dT(E&X)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT(EcoRI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT(XbaI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7.8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120719 Ag43 cut with E&amp;S(each enzyme 0.1ul) and Ag43 cut with E&amp;S(each enzyme 1ul).jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed Ag43(K346007) was successfully digested into two fragments and low concentration of restriction enzyme cause unperfectly cut of plasmid extraction product. But there are correct two bands in Ag43 cutting result so restriction enzyme worked. And about dT digestion, XbaI worked in halfway. We thought this is because we didn't added BSA buffer into dT digestion mix.<br />
<br />
And we retried digestion of dT which was plasmid extraction and gel extracted products. Recipe was same as above(E&X).<br />
<br />
===Gel extraction===<br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 20th===<br />
<div class="hokkaidou-section"><br />
====Electrophoresis====<br />
Electrophoresis for digestion result done yesterday(dT cut with EcoRI and XbaI).<br />
Added 5 ul of EtBr and Pre-migrated for 30 min.<br />
Additionaly, to confirm the concentration of Ag43(K346007) gel extract result.<br />
[[image:HokkaidoU2012 120720 dTd- dTd+ Ag43(cut with E&amp;S)gelextract.jpg|thumb|Digestion result]]<br />
From this result, we confirmed dT was successfully cut with EcoRI & SpeI.<br />
<br />
====Gel extraction====<br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
<br />
====Ethanol precipitation====<br />
Ethanol precipitation for gel extraction products above.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 5 ul of DW.<br />
<br />
====Ligation====<br />
We ligated Ag43(purified at 7/17 and 7/20) as an insert and dT as vector.<br />
<br /><br /><br />
Ag43(7/20) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|2 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ag43(7/17) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|4 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 21st===<br />
<div class="hokkaidou-section"><br />
====Electrophoresis====<br />
Electrophoresis for digestion and ligation products yesterday.<br />
<br />
[[image:HokkaidoU2012 120721 ag43-d+ dt-d+ ligation print.jpg|thumb|Digestion and Ligation results]]<br />
<br />
There are three bands in ligation products(Ag43(7/20) + dT(on pSB1AK3))). Lower band would be digestion result which couldn't be ligated with dT. Middle band would be successfully ligaed DNA which have about 6k bp(Ag43 has 3.1k bp and dT on pSB1AK3 has 3.2k bp). And higher band would make something dimer, we thought. <br />
<br />
====Single colony isolation====<br />
Single colony isolation for incubated colonies spread yesterday. <br />
<br />
====Ethanol precipitation====<br />
Ethanol precipitation for gel extracted Ag43(E&S) gel extraction product for digestion of PstI Star activity confirmation.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.<br />
<br />
====Electrophoresis====<br />
Electrophoresis to confirm the concentration of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) Ethanol precipitation for PstItest.jpg|thumb|Ethanol precipitation result]]<br />
<br />
====Digestion====<br />
Digestion to confirm there are some PstI cutting site in Ag43(K346007)(cut with EcoRI and SpeI to remove PstI this parts potentially has).<br />
<br />
{|<br />
|Used DNA <br />
|K346007 cut with EcoRI and SpeI<br />
|-<br />
|Concentration(ng/ul)<br />
|25<br />
|-<br />
|Used DNA volume(ul) <br />
|2<br />
|-<br />
|theoretical ez value(ul)<br />
|0.02<br />
|}<br />
<br />
<br />
PstI = 0.1ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.9 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.2 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.2 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.8 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.5 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 1.0 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) cut with PstI(0.1, 0.2, 0.5, 1.jpg|thumb|Digestion result]]<br />
<br />
From this digestion, we confirmed there is at least one PstI cutting site in Ag43 fragment because these digestion results showed same bp and same number of cutting band.<br />
<br />
====PCR====<br />
PCR to confirm how ligated Ag43 fragment(s) with dT on pSB1AK3.<br />
We used Ag43 last 700bp area as primer which had designed as sequencing primer. '''If three (two forward and one reverse )Ag43 were inserted, this primer can anneal to forward Ag43 as forward primer and reverse Ag43 as reverse primer then amplify about 766bp.''' If there are no reverse Ag43, DNA can't be amplified. <br />
<br /><br /><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
We failed amplification. <br />
Retry!<br />
<br />
====PCR====<br />
We did PCR written above once again. Change some point of reaction.<br />
We amplified Ag43(K346007) original DNA also as a control.<br /><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 22nd===<br />
<div class="hokkaidou-section"><br />
====Electrophoresis====<br />
Results of digestion at 21st.<br /><br />
We expected to appear the band of about 700bp.<br />
So, we use 1% and 2% agarose gel.<br />
<br />
[[image:HokkaidoU_2012_120722_ag43_pcr-1%_print-1.jpg|thumb|PCR result image]]<br />
[[image:HokkaidoU2012_120722_ag43_pcr-2%_print.jpg|thumb|PCR result image]]<br />
<br /><br />
We can't find some band in 700~800bp...<br />
<br />
====Liquid Culture====<br />
Liquid culture for single colony isolated Ag43-dT on pSB1AT3.<br />
#Picked up one colony from single colony isolated platesãby platinum loop.<br />
#Dipped into 2 ml of LBA.<br />
#Incubated for 16 hrs.<br />
<br />
====Digestion====<br />
Digested Ag43(K346oo7) plasmid extraction product with EcoRI and SpeI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_protocols
Team:HokkaidoU Japan/Notebook/plastic protocols
2012-09-27T03:56:40Z
<p>Slecat: /* Preparation for GC/MS */</p>
<hr />
<div>'''Bold text'''{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==PHB Protocols==<br />
<br />
===Polymer producing media===<br />
<div class="hokkaidou-section"><br />
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|2x LB<br />
|10 ml<br />
|-<br />
|50% Glucose<br />
|800 ul<br />
|-<br />
|1 M pantothenic acid Ca<br />
|200 ul<br />
|-<br />
|Amp(100 mg/ml)<br />
|20 ul<br />
|-<br />
|RO water (autoclaved)<br />
|8.98 ml<br />
|-<br />
|}<br />
<br />
50% gulcose (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|Glucose<br />
|10 g<br />
|-<br />
|RO water<br />
|up to 20 ml<br />
|-<br />
|}<br />
<br />
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|pantothenic acid Ca<br />
|4.77 g<br />
|-<br />
|RO water<br />
|up to 10 ml<br />
|-<br />
|}<br />
</div><br />
<br />
===Culture and harvest===<br />
<div class="hokkaidou-section"><br />
# Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.<br />
# Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.<br />
# Centrifuge for 10 min, 5,000 rpm.<br />
# Remove supernatant and add 500 ml RO water and suspend it.<br />
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.<br />
# Freeze in -80C for more than 3 hrs.<br />
# Freeze-dry for more than 48 hrs.<br />
</div><br />
<br />
===Polymer extraction and purification===<br />
<div class="hokkaidou-section"><br />
# Move dried up bacteria into test tube.<br />
# Break up them to separate and add 10 ml chloroform.<br />
# Incubate for 48 hrs at 60C.<br />
# Make it filtered through PTFE and move it into another test tube.<br />
# Volatilize organic solvent by exposing air and separate polymer.<br />
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.<br />
# Volatilize chloroform by exposing air again.<br />
# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.<br />
</div><br />
<br />
===Preparation for GC/MS===<br />
<div class="hokkaidou-section"><br />
Mixture for hydrolysis<br /><br />
All operation must be done with bare hand, so put gloves on.<br /><br />
1. Mix each solution in centrifugation tube (10 ml).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Sample<br />
|250 ul<br />
|-<br />
|HCl<br />
|100 ul<br />
|-<br />
|Ethanol<br />
|850 ul<br />
|-<br />
|}<br />
<br />
2. Voltex.<br /><br />
3. Heat at 100C for 4 hrs (each 30 min voltex).<br /><br />
4. Cool down centrifugation tube in ice.<br /><br />
5. Add solutions as follow.<br /><br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|(1)<br />
|-<br />
|0.65M NaOH<br />
|<br />
|-<br />
|0.9M NaCl<br />
|<br />
|-<br />
|<br />
|1 ml<br />
|-<br />
|(2)<br />
|-<br />
|250mM Na2HPO4 <br />
|-<br />
|(store at 4C)<br />
| 500 ul<br />
|-<br />
|}<br />
<br />
6. Voltex for 1 min.<br /><br />
7. Test by pH test paper (about pH 7.0).<br /><br />
8. Centrifugation for 5 min at 1,500 rpm.<br /><br />
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. <br />
It means the solution is passed on simple column (Dehydration).<br />
Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br /><br />
10. Remove 100 ul solution by pipetman.<br /><br />
11. Supply to GC/MS.<br /><br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_protocols
Team:HokkaidoU Japan/Notebook/plastic protocols
2012-09-27T03:55:27Z
<p>Slecat: </p>
<hr />
<div>'''Bold text'''{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==PHB Protocols==<br />
<br />
===Polymer producing media===<br />
<div class="hokkaidou-section"><br />
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|2x LB<br />
|10 ml<br />
|-<br />
|50% Glucose<br />
|800 ul<br />
|-<br />
|1 M pantothenic acid Ca<br />
|200 ul<br />
|-<br />
|Amp(100 mg/ml)<br />
|20 ul<br />
|-<br />
|RO water (autoclaved)<br />
|8.98 ml<br />
|-<br />
|}<br />
<br />
50% gulcose (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|Glucose<br />
|10 g<br />
|-<br />
|RO water<br />
|up to 20 ml<br />
|-<br />
|}<br />
<br />
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|pantothenic acid Ca<br />
|4.77 g<br />
|-<br />
|RO water<br />
|up to 10 ml<br />
|-<br />
|}<br />
</div><br />
<br />
===Culture and harvest===<br />
<div class="hokkaidou-section"><br />
# Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.<br />
# Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.<br />
# Centrifuge for 10 min, 5,000 rpm.<br />
# Remove supernatant and add 500 ml RO water and suspend it.<br />
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.<br />
# Freeze in -80C for more than 3 hrs.<br />
# Freeze-dry for more than 48 hrs.<br />
</div><br />
<br />
===Polymer extraction and purification===<br />
<div class="hokkaidou-section"><br />
# Move dried up bacteria into test tube.<br />
# Break up them to separate and add 10 ml chloroform.<br />
# Incubate for 48 hrs at 60C.<br />
# Make it filtered through PTFE and move it into another test tube.<br />
# Volatilize organic solvent by exposing air and separate polymer.<br />
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.<br />
# Volatilize chloroform by exposing air again.<br />
# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.<br />
</div><br />
<br />
===Preparation for GC/MS===<br />
<div class="hokkaidou-section"><br />
Mixture for hydrolysis<br /><br />
All operation must be done with bare hand, so put gloves on.<br /><br />
1. Mix each solution in centrifugation tube (10 ml).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Sample<br />
|250 ul<br />
|-<br />
|HCl<br />
|100 ul<br />
|-<br />
|Ethanol<br />
|850 ul<br />
|-<br />
|}<br />
<br />
2. Voltex.<br /><br />
3. Heat at 100C for 4 hrs (each 30 min voltex).<br /><br />
4. Cool down centrifugation tube in ice.<br /><br />
5. Add solutions as follow.<br /><br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|(1)<br />
|-<br />
|0.65M NaOH<br />
|<br />
|-<br />
|0.9M NaCl<br />
|<br />
|-<br />
|<br />
|1 ml<br />
|-<br />
|(2)<br />
|-<br />
|250mM Na2HPO4 <br />
|-<br />
|(store at 4C)<br />
| 500 ul<br />
|-<br />
|}<br />
<br />
6. Voltex for 1 min.<br /><br />
7. Test by pH test paper (about pH 7.0).<br /><br />
8. Centrifugation for 5 min at 1,500rpm.<br /><br />
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. <br />
It means the solution is passed on simple column (Dehydration).<br />
Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br /><br />
10. Remove 100 ul solution by pipetman.<br /><br />
11. Supply to GC/MS.<br /><br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_protocols
Team:HokkaidoU Japan/Notebook/plastic protocols
2012-09-27T03:54:32Z
<p>Slecat: /* Preparation for GC/MS */</p>
<hr />
<div>'''Bold text'''{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==PHB Protocols==<br />
<br />
===Polymer producing media===<br />
<div class="hokkaidou-section"><br />
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|2x LB<br />
|10 ml<br />
|-<br />
|50% Glucose<br />
|800 ul<br />
|-<br />
|1 M pantothenic acid Ca<br />
|200 ul<br />
|-<br />
|Amp(100 mg/ml)<br />
|20 ul<br />
|-<br />
|RO water (autoclaved)<br />
|8.98 ml<br />
|-<br />
|}<br />
<br />
50% gulcose (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|Glucose<br />
|10 g<br />
|-<br />
|RO water<br />
|up to 20 ml<br />
|-<br />
|}<br />
<br />
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|pantothenic acid Ca<br />
|4.77 g<br />
|-<br />
|RO water<br />
|up to 10 ml<br />
|-<br />
|}<br />
</div><br />
<br />
===Culture and harvest===<br />
<div class="hokkaidou-section"><br />
# Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.<br />
# Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.<br />
# Centrifuge for 10 min, 5,000 rpm.<br />
# Remove supernatant and add 500 ml RO water and suspend it.<br />
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.<br />
# Freeze in -80C for more than 3 hrs.<br />
# Freeze-dry for more than 48 hrs.<br />
</div><br />
<br />
===Polymer extraction and purification===<br />
<div class="hokkaidou-section"><br />
# Move dried up bacteria into test tube.<br />
# Break up them to separate and add 10 ml chloroform.<br />
# Incubate for 48 hrs at 60C.<br />
# Make it filtered through PTFE and move it into another test tube.<br />
# Volatilize organic solvent by exposing air and separate polymer.<br />
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.<br />
# Volatilize chloroform by exposing air again.<br />
# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.<br />
</div><br />
<br />
===Preparation for GC/MS===<br />
<div class="hokkaidou-section"><br />
Mixture for hydrolysis<br><br />
All operation must be done with bare hand, so put gloves on.<br><br />
1. Mix each solution in centrifugation tube (10 ml).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Sample<br />
|250 ul<br />
|-<br />
|HCl<br />
|100 ul<br />
|-<br />
|Ethanol<br />
|850 ul<br />
|-<br />
|}<br />
<br />
2. Voltex.<br><br />
3. Heat at 100C for 4 hrs (each 30 min voltex).<br><br />
4. Cool down centrifugation tube in ice.<br><br />
5. Add solutions as follow.<br><br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|(1)<br />
|-<br />
|0.65M NaOH<br />
|<br />
|-<br />
|0.9M NaCl<br />
|<br />
|-<br />
|<br />
|1 ml<br />
|-<br />
|(2)<br />
|-<br />
|250mM Na2HPO4 <br />
|-<br />
|(store at 4C)<br />
| 500 ul<br />
|-<br />
|}<br />
<br />
6. Voltex for 1 min.<br><br />
7. Test by pH test paper (about pH 7.0).<br><br />
8. Centrifugation for 5 min at 1,500rpm.<br><br />
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. <br />
It means the solution is passed on simple column (Dehydration).<br />
Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br><br />
10. Remove 100 ul solution by pipetman.<br><br />
11. Supply to GC/MS.<br><br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_protocols
Team:HokkaidoU Japan/Notebook/plastic protocols
2012-09-27T03:54:08Z
<p>Slecat: </p>
<hr />
<div>'''Bold text'''{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==PHB Protocols==<br />
<br />
===Polymer producing media===<br />
<div class="hokkaidou-section"><br />
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|2x LB<br />
|10 ml<br />
|-<br />
|50% Glucose<br />
|800 ul<br />
|-<br />
|1 M pantothenic acid Ca<br />
|200 ul<br />
|-<br />
|Amp(100 mg/ml)<br />
|20 ul<br />
|-<br />
|RO water (autoclaved)<br />
|8.98 ml<br />
|-<br />
|}<br />
<br />
50% gulcose (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|Glucose<br />
|10 g<br />
|-<br />
|RO water<br />
|up to 20 ml<br />
|-<br />
|}<br />
<br />
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|pantothenic acid Ca<br />
|4.77 g<br />
|-<br />
|RO water<br />
|up to 10 ml<br />
|-<br />
|}<br />
</div><br />
<br />
===Culture and harvest===<br />
<div class="hokkaidou-section"><br />
# Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.<br />
# Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.<br />
# Centrifuge for 10 min, 5,000 rpm.<br />
# Remove supernatant and add 500 ml RO water and suspend it.<br />
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.<br />
# Freeze in -80C for more than 3 hrs.<br />
# Freeze-dry for more than 48 hrs.<br />
</div><br />
<br />
===Polymer extraction and purification===<br />
<div class="hokkaidou-section"><br />
# Move dried up bacteria into test tube.<br />
# Break up them to separate and add 10 ml chloroform.<br />
# Incubate for 48 hrs at 60C.<br />
# Make it filtered through PTFE and move it into another test tube.<br />
# Volatilize organic solvent by exposing air and separate polymer.<br />
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.<br />
# Volatilize chloroform by exposing air again.<br />
# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.<br />
</div><br />
<br />
===Preparation for GC/MS===<br />
<div class="hokkaidou-section"><br />
Mixture for hydrolysis<br><br />
All operation must be done with bare hand, so put gloves on.<br><br />
1. Mix each solution in centrifugation tube (10ml).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Sample<br />
|250 ul<br />
|-<br />
|HCl<br />
|100 ul<br />
|-<br />
|Ethanol<br />
|850 ul<br />
|-<br />
|}<br />
<br />
2. Voltex.<br><br />
3. Heat at 100C for 4 hrs (each 30 min voltex).<br><br />
4. Cool down centrifugation tube in ice.<br><br />
5. Add solutions as follow.<br><br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|(1)<br />
|-<br />
|0.65M NaOH<br />
|<br />
|-<br />
|0.9M NaCl<br />
|<br />
|-<br />
|<br />
|1 ml<br />
|-<br />
|(2)<br />
|-<br />
|250mM Na2HPO4 <br />
|-<br />
|(store at 4C)<br />
| 500 ul<br />
|-<br />
|}<br />
<br />
6. Voltex for 1 min.<br><br />
7. Test by pH test paper (about pH 7.0).<br><br />
8. Centrifugation for 5 min at 1,500rpm.<br><br />
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. <br />
It means the solution is passed on simple column (Dehydration).<br />
Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br><br />
10. Remove 100 ul solution by pipetman.<br><br />
11. Supply to GC/MS.<br><br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_protocols
Team:HokkaidoU Japan/Notebook/plastic protocols
2012-09-27T03:53:13Z
<p>Slecat: /* Polymer extraction and purification */</p>
<hr />
<div>'''Bold text'''{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==PHB Protocols==<br />
<br />
===Polymer producing media===<br />
<div class="hokkaidou-section"><br />
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|2x LB<br />
|10 ml<br />
|-<br />
|50% Glucose<br />
|800 ul<br />
|-<br />
|1 M pantothenic acid Ca<br />
|200 ul<br />
|-<br />
|Amp(100 mg/ml)<br />
|20 ul<br />
|-<br />
|RO water (autoclaved)<br />
|8.98 ml<br />
|-<br />
|}<br />
<br />
50% gulcose (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|Glucose<br />
|10 g<br />
|-<br />
|RO water<br />
|up to 20 ml<br />
|-<br />
|}<br />
<br />
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|pantothenic acid Ca<br />
|4.77 g<br />
|-<br />
|RO water<br />
|up to 10 ml<br />
|-<br />
|}<br />
</div><br />
<br />
===Culture and harvest===<br />
<div class="hokkaidou-section"><br />
# Preculture transformed media 1.5 ml for 10~14 hours, 180 rpm/ 30C.<br />
# Culture 15 ul preculture media in polymer producing media for 48 hours, 180 rpm/ 30C.<br />
# Centrifuge for 10 min, 5,000 rpm.<br />
# Remove supernatant and add 500 ml RO water and suspend it.<br />
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.<br />
# Freeze in -80C for more than 3 hours.<br />
# Freeze-dry for more than 48 hours.<br />
</div><br />
<br />
===Polymer extraction and purification===<br />
<div class="hokkaidou-section"><br />
# Move dried up bacteria into test tube.<br />
# Break up them to separate and add 10 ml chloroform.<br />
# Incubate for 48 hrs at 60C.<br />
# Make it filtered through PTFE and move it into another test tube.<br />
# Volatilize organic solvent by exposing air and separate polymer.<br />
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.<br />
# Volatilize chloroform by exposing air again.<br />
# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.<br />
</div><br />
<br />
===Preparation for GC/MS===<br />
<div class="hokkaidou-section"><br />
Mixture for hydrolysis<br><br />
All operation must be done with bare hand, so put gloves on.<br><br />
1. Mix each solution in centrifugation tube (10ml).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Sample<br />
|250 ul<br />
|-<br />
|HCl<br />
|100 ul<br />
|-<br />
|Ethanol<br />
|850 ul<br />
|-<br />
|}<br />
<br />
2. Voltex.<br><br />
3. Heat at 100C for 4 hours (each 30 min voltex).<br><br />
4. Cool down centrifugation tube in ice.<br><br />
5. Add solutions as follow.<br><br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|(1)<br />
|-<br />
|0.65M NaOH<br />
|<br />
|-<br />
|0.9M NaCl<br />
|<br />
|-<br />
|<br />
|1 ml<br />
|-<br />
|(2)<br />
|-<br />
|250mM Na2HPO4 <br />
|-<br />
|(store at 4C)<br />
| 500 ul<br />
|-<br />
|}<br />
<br />
6. Voltex for 1 min.<br><br />
7. Test by pH test paper (about pH 7.0).<br><br />
8. Centrifugation for 5 min at 1,500rpm.<br><br />
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. <br />
It means the solution is passed on simple column (Dehydration).<br />
Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br><br />
10. Remove 100 ul solution by pipetman.<br><br />
11. Supply to GC/MS.<br><br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_protocols
Team:HokkaidoU Japan/Notebook/plastic protocols
2012-09-27T03:52:30Z
<p>Slecat: /* Polymer producing media */</p>
<hr />
<div>'''Bold text'''{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==PHB Protocols==<br />
<br />
===Polymer producing media===<br />
<div class="hokkaidou-section"><br />
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|2x LB<br />
|10 ml<br />
|-<br />
|50% Glucose<br />
|800 ul<br />
|-<br />
|1 M pantothenic acid Ca<br />
|200 ul<br />
|-<br />
|Amp(100 mg/ml)<br />
|20 ul<br />
|-<br />
|RO water (autoclaved)<br />
|8.98 ml<br />
|-<br />
|}<br />
<br />
50% gulcose (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|Glucose<br />
|10 g<br />
|-<br />
|RO water<br />
|up to 20 ml<br />
|-<br />
|}<br />
<br />
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|RO water<br />
|7 ml<br />
|-<br />
|pantothenic acid Ca<br />
|4.77 g<br />
|-<br />
|RO water<br />
|up to 10 ml<br />
|-<br />
|}<br />
</div><br />
<br />
===Culture and harvest===<br />
<div class="hokkaidou-section"><br />
# Preculture transformed media 1.5 ml for 10~14 hours, 180 rpm/ 30C.<br />
# Culture 15 ul preculture media in polymer producing media for 48 hours, 180 rpm/ 30C.<br />
# Centrifuge for 10 min, 5,000 rpm.<br />
# Remove supernatant and add 500 ml RO water and suspend it.<br />
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.<br />
# Freeze in -80C for more than 3 hours.<br />
# Freeze-dry for more than 48 hours.<br />
</div><br />
<br />
===Polymer extraction and purification===<br />
<div class="hokkaidou-section"><br />
# Move dried up bacteria into test tube.<br />
# Break up them to separate and add 10 ml chloroform.<br />
# Incubate for 48 hour at 60C.<br />
# Make it filtered through PTFE and move it into another test tube.<br />
# Volatilize organic solvent by exposing air and separate polymer.<br />
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.<br />
# Volatilize chloroform by exposing air again.<br />
# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.<br />
</div><br />
<br />
===Preparation for GC/MS===<br />
<div class="hokkaidou-section"><br />
Mixture for hydrolysis<br><br />
All operation must be done with bare hand, so put gloves on.<br><br />
1. Mix each solution in centrifugation tube (10ml).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Sample<br />
|250 ul<br />
|-<br />
|HCl<br />
|100 ul<br />
|-<br />
|Ethanol<br />
|850 ul<br />
|-<br />
|}<br />
<br />
2. Voltex.<br><br />
3. Heat at 100C for 4 hours (each 30 min voltex).<br><br />
4. Cool down centrifugation tube in ice.<br><br />
5. Add solutions as follow.<br><br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|(1)<br />
|-<br />
|0.65M NaOH<br />
|<br />
|-<br />
|0.9M NaCl<br />
|<br />
|-<br />
|<br />
|1 ml<br />
|-<br />
|(2)<br />
|-<br />
|250mM Na2HPO4 <br />
|-<br />
|(store at 4C)<br />
| 500 ul<br />
|-<br />
|}<br />
<br />
6. Voltex for 1 min.<br><br />
7. Test by pH test paper (about pH 7.0).<br><br />
8. Centrifugation for 5 min at 1,500rpm.<br><br />
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. <br />
It means the solution is passed on simple column (Dehydration).<br />
Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br><br />
10. Remove 100 ul solution by pipetman.<br><br />
11. Supply to GC/MS.<br><br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:48:57Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==Introduction==<br />
<div class="hokkaidou-section"><br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
</div><br />
==Method==<br />
<div class="hokkaidou-section"><br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|650px|Fig. 1 Image of our Bio capsule]]<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br />
</p><br />
</div><br />
<br />
==Results==<br />
<div class="hokkaidou-section"><br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates to start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.</p><br />
[[image:HokkaidoU Table1.jpg|center|thumb|650px|Table1]]<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|650px|fig. 2]]<br />
<p><br />
Fig. 2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in fig. 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in fig. 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig. 3).<br />
</p><br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|650px|Fig. 3 clustered cells]]<br />
<div style="width: 100%; height: 30px;"></div><br />
====precipitation test====<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
</p><br />
<div style="width: 100%; height: 30px;"></div><br />
====Staining by Nile red====<br />
<p><br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red and P3HB was detected only in the presence of plastic producing module(right panel above), but not in the absence of it(left panel above)<br />
</p> <br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|Staining with Nile red (Fluorescenced photograph above)]]<br />
<div style="width: 100%; height: 30px;"></div><br />
====HPLC test====<br />
<p><br />
We confirmed the existence of P3HB by analyzing building block of it after hydrolysis of P3HB by using HPLC.<br />
Degraded building block of P3HB was detected in E. coli containing both "plastic producing module" and "Aggregation module".<br />
</p><br />
[[image:pGEM+Ag (A G AG).jpg|center|thumb|350px|Result of HPLC test, A=added only arabinose G=added only glucose AG=addded both arabinose and glucose.]]<br />
</div><br />
<br />
==Discussion(Improvement)==<br />
<div class="hokkaidou-section"><br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
[[image:HokkaidoU Ag43+PHB もっと強くなる!.png|center|thumb|1000px|Fig. 4 image]]<br />
</div><br />
==Future planning==<br />
<div class="hokkaidou-section"><br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
</div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis
Team:HokkaidoU Japan/Project/PHB Synthesis
2012-09-27T03:46:30Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==Planning==<br />
<div class="hokkaidou-section"><br />
<p>We attempted to make ''Escherichia coli'' producing bio-plastic for demonstrating industrial applicability of “Aggregation Module” that makes “bio-capsules” formed by aggregation of ''E. coli'' through cell-cell interaction via adhesion molecule, Ag43, located on the surface of outer membrane.</p><br />
<p>Development of a cost-effective method for manufacturing biodegradable plastic is one of important issues for making sustainable future society. Common plastic is made from oil (Fig. 1). Oil is limited resource, since it is estimated to be exhausted just in 46.2 years<sup>[1]</sup>. Synthetic plastic also cause problem as a stable waste material since it is not bio-degradable. Bio-Plastic will never contribute to the increase of atmospheric carbon dioxide since bacteria utilizes glucose as resource for its biosynthesis (Fig. 2).</p><br />
[[image:HokkaidoU_PHB_Fig1.jpg|316px|left|thumb|Fig. 1. The problem of common plastic.]][[image:HokkaidoU_PHB_Fig2.jpg|302px|right|thumb|Fig. 2. Bio-Plastic is eco-friendly.]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Background==<br />
<div class="hokkaidou-section"><br />
<p>Poly-3-hydroxy-butyrate P(3HB) is one of bio-plastics made by bacteria and archaea. They store P(3HB) in their cells as energy-storage molecule<sup>[2]</sup>. So bio-plastic is bio-degradable. Those bacteria synthesize it from acetyl-CoA, the intermediate product of glycolysis. Monomer of P(3HB) is also used as material for making several other useful plastics (co-polymer) through coupling with other types of component molecules like valeric acid or lactic acid<sup>[3]</sup>. <br />
''Ralstonia eutropha'' is well-known as hydrogen bacteria that produce P(3HB).</p><br />
<p>In ''R. eutropha'' cells, P(3HB) is made through 3 steps (Fig. 3). Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB)<sup>[2]</sup>. </p><br />
[[image:HokkaidoU_PHB_Fig3.jpg|673px|thumb|Fig. 3. P(3HB) synthesis pathway in ''R. eutropha''.]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Experiments==<br />
<div class="hokkaidou-section"><br />
====Optimization of P(3HB) production====<br />
<p>We tried optimization of P(3HB) production under four conditions: ''E. coli'' host strain, addition of pantothenic acid (PA), Glucose concentration and liquid culture mediun. P(3HB) wad produced by using pGEM(phaCAB) that covers all region need for P(3HB) biosynthesis found in ''R. eutropha'' genome, which was provided by Taguchi Lab.</p><br />
====Construction of P(3HB) producing module====<br />
<p>We constructed P(3HB) producing module (Fig. 4). Genes encoding PhaA and PhaB were obtained by subcloning from pGEM(phaCAB). PhaC has already registered as BBa_K342001 by INSA-Lyon2010. We appreciate INSA-Lyon 2010 teams effort. We ligated these genes in the order of PhaC, PhaA, PhaB for making BioBrick of “Plastic Producing Module”. TetR repressible promoter (BBa_R0040 ) and RBS (BBa_B0034) are fused to upstream of above gene cluster. </p><br />
[[image:HokkaidoU_PHB_Fig4.jpg|673px|thumb|Fig. 4. Construction of P(3HB) producing module]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Results==<br />
<div class="hokkaidou-section"><br />
====Optimization of P(3HB) production====<br />
[[image:HokkaidoU_PHB_Fig5.jpg|673px|thumb|Fig. 5. Production of P(3HB) under various conditions.]]<br />
<p>P(3HB) production was drastically changed in several conditions (Fig. 5). “Plastic producing module” in JM109 have shown more efficient plastic production than that in DH5α (1.45 fold). In the same condition, concentration of glucose and presence of PA affects the yield of plastic production. We found that TB medium, rich medium, produced 1.64 fold more plastic than LB medium.</p><br />
====Construction of P(3HB) producing module====<br />
<p>We did not succeed in producing plastic by culturing JM109/pSB(phaCAB) containing BioBrick part in pSB1C3 yet. Now we are checking several culture condition and promoter species for controlling their gene expression.</p><br />
</div><br />
<br />
==Discussion==<br />
<div class="hokkaidou-section"><br />
<p>We succeeded in optimization of plastic production using pGEM(phaCAB), however, pSB(phaCAB) did not work as we expected. ''E. coli'' which was transformed with pGEM(phaCAB) produced plenty of PHB, so that our method of culturing and producing PHB works well. As for the reason why PHB was not produced with our phaCAB BioBrick part, we think that an over expression of phaCAB genes caused the resulting enzymes being transferred into inclusion bodies. We assure that ''E. coli'' which is transformed pGEM(phaCAB) produces P(3HB) because pGEM(phaCAB) includes phaCAB operon which derived from ''R. eutropha'' genome. However, in our experiment, we used PhaC (BBa_K342001, INSA-Lyon2010) which has codon optimization to be fit for translation of ''E. coli''. Furthermore, RBS and promoter which we used are ''E. coli'' genes. TetR repressible promoter (BBa_R0040 ) is stronger than a promoter on pGEM(phaCAB). In this case, phaCAB genes may be expressed more in ''E. coli'' than in the case of pGEM(phaCAB).</p><br />
<p>To confirm our hypothesis, we should examine whether the intermediates are produced. The data that two intermediates of P(3HB) producing pathway are synthesized but not the final product should mean that the enzymes are put in inclusion bodies. In addition, to make our phaCAB BioBrick part better we will replace TetR repressible promoter with pGEM(phaCAB) promoter. We guess that due to the pGEM(phaCAB) promoter the expression of phaCAB genes will be less than before, and PHB is produced successfully.</p><br />
<p>We hope many iGEM teams will be able to produce plastic easily with our parts and they will make future iGEM projects more eco-friendly and more attractive.</p><br />
</div><br />
<br />
==References==<br />
<div class="hokkaidou-section"><br />
# H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010)<br />
# Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006).<br />
# 田口精一: バイオプラスチックのつくられ方とつくり方 蛋白質 核酸 酵素,50.3: 262-269 (2005) (in Japanese)<br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis
Team:HokkaidoU Japan/Project/PHB Synthesis
2012-09-27T03:45:05Z
<p>Slecat: /* Discussion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==Planning==<br />
<div class="hokkaidou-section"><br />
<p>We attempted to make ''Escherichia coli'' producing bio-plastic for demonstrating industrial applicability of “Aggregation Module” that makes “bio-capsules” formed by aggregation of ''E. coli'' through cell-cell interaction via adhesion molecule, Ag43, located on the surface of outer membrane.</p><br />
<p>Development of a cost-effective method for manufacturing biodegradable plastic is one of important issues for making sustainable future society. Common plastic is made from oil (Figure 1). Oil is limited resource, since it is estimated to be exhausted just in 46.2 years<sup>[1]</sup>. Synthetic plastic also cause problem as a stable waste material since it is not bio-degradable. Bio-Plastic will never contribute to the increase of atmospheric carbon dioxide since bacteria utilizes glucose as resource for its biosynthesis (Figure 2).</p><br />
[[image:HokkaidoU_PHB_Fig1.jpg|316px|left|thumb|Figure 1. The problem of common plastic.]][[image:HokkaidoU_PHB_Fig2.jpg|302px|right|thumb|Figure 2. Bio-Plastic is eco-friendly.]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Background==<br />
<div class="hokkaidou-section"><br />
<p>Poly-3-hydroxy-butyrate P(3HB) is one of bio-plastics made by bacteria and archaea. They store P(3HB) in their cells as energy-storage molecule<sup>[2]</sup>. So bio-plastic is bio-degradable. Those bacteria synthesize it from acetyl-CoA, the intermediate product of glycolysis. Monomer of P(3HB) is also used as material for making several other useful plastics (co-polymer) through coupling with other types of component molecules like valeric acid or lactic acid<sup>[3]</sup>. <br />
''Ralstonia eutropha'' is well-known as hydrogen bacteria that produce P(3HB).</p><br />
<p>In ''R. eutropha'' cells, P(3HB) is made through 3 steps (Figure 3). Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB)<sup>[2]</sup>. </p><br />
[[image:HokkaidoU_PHB_Fig3.jpg|673px|thumb|Figure 3. P(3HB) synthesis pathway in ''R. eutropha''.]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Experiments==<br />
<div class="hokkaidou-section"><br />
====Optimization of P(3HB) production====<br />
<p>We tried optimization of P(3HB) production under four conditions: ''E. coli'' host strain, addition of pantothenic acid (PA), Glucose concentration and liquid culture mediun. P(3HB) wad produced by using pGEM(phaCAB) that covers all region need for P(3HB) biosynthesis found in ''R. eutropha'' genome, which was provided by Taguchi Lab.</p><br />
====Construction of P(3HB) producing module====<br />
<p>We constructed P(3HB) producing module (Figure 4). Genes encoding PhaA and PhaB were obtained by subcloning from pGEM(phaCAB). PhaC has already registered as BBa_K342001 by INSA-Lyon2010. We appreciate INSA-Lyon 2010 teams effort. We ligated these genes in the order of PhaC, PhaA, PhaB for making BioBrick of “Plastic Producing Module”. TetR repressible promoter (BBa_R0040 ) and RBS (BBa_B0034) are fused to upstream of above gene cluster. </p><br />
[[image:HokkaidoU_PHB_Fig4.jpg|673px|thumb|Figure 4. Construction of P(3HB) producing module]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Results==<br />
<div class="hokkaidou-section"><br />
====Optimization of P(3HB) production====<br />
[[image:HokkaidoU_PHB_Fig5.jpg|673px|thumb|Figure 5. Production of P(3HB) under various conditions.]]<br />
<p>P(3HB) production was drastically changed in several conditions (Figure 5). “Plastic producing module” in JM109 have shown more efficient plastic production than that in DH5α (1.45 fold). In the same condition, concentration of glucose and presence of PA affects the yield of plastic production. We found that TB medium, rich medium, produced 1.64 fold more plastic than LB medium.</p><br />
====Construction of P(3HB) producing module====<br />
<p>We did not succeed in producing plastic by culturing JM109/pSB(phaCAB) containing BioBrick part in pSB1C3 yet. Now we are checking several culture condition and promoter species for controlling their gene expression.</p><br />
</div><br />
<br />
==Discussion==<br />
<div class="hokkaidou-section"><br />
<p>We succeeded in optimization of plastic production using pGEM(phaCAB), however, pSB(phaCAB) did not work as we expected. ''E. coli'' which was transformed with pGEM(phaCAB) produced plenty of PHB, so that our method of culturing and producing PHB works well. As for the reason why PHB was not produced with our phaCAB BioBrick part, we think that an over expression of phaCAB genes caused the resulting enzymes being transferred into inclusion bodies. We assure that ''E. coli'' which is transformed pGEM(phaCAB) produces P(3HB) because pGEM(phaCAB) includes phaCAB operon which derived from ''R. eutropha'' genome. However, in our experiment, we used PhaC (BBa_K342001, INSA-Lyon2010) which has codon optimization to be fit for translation of ''E. coli''. Furthermore, RBS and promoter which we used are ''E. coli'' genes. TetR repressible promoter (BBa_R0040 ) is stronger than a promoter on pGEM(phaCAB). In this case, phaCAB genes may be expressed more in ''E. coli'' than in the case of pGEM(phaCAB).</p><br />
<p>To confirm our hypothesis, we should examine whether the intermediates are produced. The data that two intermediates of P(3HB) producing pathway are synthesized but not the final product should mean that the enzymes are put in inclusion bodies. In addition, to make our phaCAB BioBrick part better we will replace TetR repressible promoter with pGEM(phaCAB) promoter. We guess that due to the pGEM(phaCAB) promoter the expression of phaCAB genes will be less than before, and PHB is produced successfully.</p><br />
<p>We hope many iGEM teams will be able to produce plastic easily with our parts and they will make future iGEM projects more eco-friendly and more attractive.</p><br />
</div><br />
<br />
==References==<br />
<div class="hokkaidou-section"><br />
# H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010)<br />
# Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006).<br />
# 田口精一: バイオプラスチックのつくられ方とつくり方 蛋白質 核酸 酵素,50.3: 262-269 (2005) (in Japanese)<br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis
Team:HokkaidoU Japan/Project/PHB Synthesis
2012-09-27T03:44:04Z
<p>Slecat: /* Discussion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
==Planning==<br />
<div class="hokkaidou-section"><br />
<p>We attempted to make ''Escherichia coli'' producing bio-plastic for demonstrating industrial applicability of “Aggregation Module” that makes “bio-capsules” formed by aggregation of ''E. coli'' through cell-cell interaction via adhesion molecule, Ag43, located on the surface of outer membrane.</p><br />
<p>Development of a cost-effective method for manufacturing biodegradable plastic is one of important issues for making sustainable future society. Common plastic is made from oil (Figure 1). Oil is limited resource, since it is estimated to be exhausted just in 46.2 years<sup>[1]</sup>. Synthetic plastic also cause problem as a stable waste material since it is not bio-degradable. Bio-Plastic will never contribute to the increase of atmospheric carbon dioxide since bacteria utilizes glucose as resource for its biosynthesis (Figure 2).</p><br />
[[image:HokkaidoU_PHB_Fig1.jpg|316px|left|thumb|Figure 1. The problem of common plastic.]][[image:HokkaidoU_PHB_Fig2.jpg|302px|right|thumb|Figure 2. Bio-Plastic is eco-friendly.]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Background==<br />
<div class="hokkaidou-section"><br />
<p>Poly-3-hydroxy-butyrate P(3HB) is one of bio-plastics made by bacteria and archaea. They store P(3HB) in their cells as energy-storage molecule<sup>[2]</sup>. So bio-plastic is bio-degradable. Those bacteria synthesize it from acetyl-CoA, the intermediate product of glycolysis. Monomer of P(3HB) is also used as material for making several other useful plastics (co-polymer) through coupling with other types of component molecules like valeric acid or lactic acid<sup>[3]</sup>. <br />
''Ralstonia eutropha'' is well-known as hydrogen bacteria that produce P(3HB).</p><br />
<p>In ''R. eutropha'' cells, P(3HB) is made through 3 steps (Figure 3). Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB)<sup>[2]</sup>. </p><br />
[[image:HokkaidoU_PHB_Fig3.jpg|673px|thumb|Figure 3. P(3HB) synthesis pathway in ''R. eutropha''.]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Experiments==<br />
<div class="hokkaidou-section"><br />
====Optimization of P(3HB) production====<br />
<p>We tried optimization of P(3HB) production under four conditions: ''E. coli'' host strain, addition of pantothenic acid (PA), Glucose concentration and liquid culture mediun. P(3HB) wad produced by using pGEM(phaCAB) that covers all region need for P(3HB) biosynthesis found in ''R. eutropha'' genome, which was provided by Taguchi Lab.</p><br />
====Construction of P(3HB) producing module====<br />
<p>We constructed P(3HB) producing module (Figure 4). Genes encoding PhaA and PhaB were obtained by subcloning from pGEM(phaCAB). PhaC has already registered as BBa_K342001 by INSA-Lyon2010. We appreciate INSA-Lyon 2010 teams effort. We ligated these genes in the order of PhaC, PhaA, PhaB for making BioBrick of “Plastic Producing Module”. TetR repressible promoter (BBa_R0040 ) and RBS (BBa_B0034) are fused to upstream of above gene cluster. </p><br />
[[image:HokkaidoU_PHB_Fig4.jpg|673px|thumb|Figure 4. Construction of P(3HB) producing module]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div><br />
<br />
==Results==<br />
<div class="hokkaidou-section"><br />
====Optimization of P(3HB) production====<br />
[[image:HokkaidoU_PHB_Fig5.jpg|673px|thumb|Figure 5. Production of P(3HB) under various conditions.]]<br />
<p>P(3HB) production was drastically changed in several conditions (Figure 5). “Plastic producing module” in JM109 have shown more efficient plastic production than that in DH5α (1.45 fold). In the same condition, concentration of glucose and presence of PA affects the yield of plastic production. We found that TB medium, rich medium, produced 1.64 fold more plastic than LB medium.</p><br />
====Construction of P(3HB) producing module====<br />
<p>We did not succeed in producing plastic by culturing JM109/pSB(phaCAB) containing BioBrick part in pSB1C3 yet. Now we are checking several culture condition and promoter species for controlling their gene expression.</p><br />
</div><br />
<br />
==Discussion==<br />
<div class="hokkaidou-section"><br />
<p>We succeeded in optimization of plastic production using pGEM(phaCAB), however, pSB(phaCAB) did not work as we expected. ''E. coli'' which was transformed with pGEM(phaCAB) produced plenty of PHB, so that our method of culturing and producing PHB works well. As for the reason why PHB was not produced with our phaCAB BioBrick part, we think that an over expression of phaCAB genes caused the resulting enzymes being transferred into inclusion bodies. We assure that ''E. coli'' which is transformed pGEM(phaCAB) produces P(3HB) because pGEM(phaCAB) includes phaCAB operon which derived from ''R. eutropha'' genome. However, in our experiment, we used PhaC (BBa_K342001, INSA-Lyon2010) which has codon optimization to be fit for translation of E. coli. Furthermore, RBS and promoter which we used are ''E. coli'' genes. TetR repressible promoter (BBa_R0040 ) is stronger than a promoter on pGEM(phaCAB). In this case, phaCAB genes may be expressed more in ''E. coli'' than in the case of pGEM(phaCAB).</p><br />
<p>To confirm our hypothesis, we should examine whether the intermediates are produced. The data that two intermediates of P(3HB) producing pathway are synthesized but not the final product should mean that the enzymes are put in inclusion bodies. In addition, to make our phaCAB BioBrick part better we will replace TetR repressible promoter with pGEM(phaCAB) promoter. We guess that due to the pGEM(phaCAB) promoter the expression of phaCAB genes will be less than before, and PHB is produced successfully.</p><br />
<p>We hope many iGEM teams will be able to produce plastic easily with our parts and they will make future iGEM projects more eco-friendly and more attractive.</p><br />
</div><br />
<br />
==References==<br />
<div class="hokkaidou-section"><br />
# H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010)<br />
# Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006).<br />
# 田口精一: バイオプラスチックのつくられ方とつくり方 蛋白質 核酸 酵素,50.3: 262-269 (2005) (in Japanese)<br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Aggregation
Team:HokkaidoU Japan/Project/Aggregation
2012-09-27T03:32:58Z
<p>Slecat: /* Experiments */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
==Planning of "Aggregation Module"==<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 Ag43 Figure1.JPG|right|thumb|300px|Fig. 1 Large amount of cultivated cells is time-consuming to harvest by centrifugation]]<br />
<p><br />
In modern industry, two major methods to harvest cultured cells that produce high-value added macromolecules are centrifugation and filtration. Since sizes of Escherichia coli cells are very small, harvesting them by filtration is difficult. So the most popular method for harvesting E. coli is centrifugation. However centrifugation is time-consuming process particularly when harvest them from large volume of culture medium. (Fig. 1)<br />
</p><br />
<p><br />
The purpose of this project is to make more efficient method for macromolecule production in bacterial cells by eliminating time-consuming centrifugation step, through enlarging sizes of E. coli cell clusters (large enough to be captured), named “Bio-capsule”, for harvesting them by filtration on nylon mesh.<br />
</p><br />
<p><br />
For this purpose, we made E. coli expressing adhesion molecule, Antigen 43 (Ag43), on the surface of outer membrane of it for enhancing cell-cell interaction. We have succeeded in harvesting large amount of E. coli cells as filter cake on nylon mesh after continuous rapid strokes of injector (Fig. 2). The “Bio-capsule” resulted from function of the “Aggregation Module” will contribute to the industrial production in near future.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Figure2.JPG|center|thumb|400px|Fig. 2 Our capturing tools and harvesting method]]<br />
</div><br />
<br />
==Background of "Aggregation Module"==<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 Ag43 Figure3 .JPG|center|thumb|500px|Fig. 3 Cell-to-cell aggregation by Ag43 complex. &alpha;43 domain exists at 10 nm above on the E. coli cell surface by non-covelent bond with &beta;43 domain (right). Left picture is actual cluster observed with optical microscopy.]]<br />
<p><br />
Ag43 is one of a kind of outer membrane proteins and belongs to auto-transporter family. This protein was derived from E. coli locus: flu, mapping on the E. coli K-12 chromosome. Other E. coli strains also have same gene, but this K-12 strain’s Ag43 cause the most rapid aggregation compared with other strain’s Ag43 [2]. Typical auto-transporter domains: signal peptides, N-proximal passenger domain (&alpha;43 : processing sites), autochaperon domain and C-terminal β-barrel domain (&beta;43 : translocation unit) are found in the protein. These domains promote cell-to-cell auto-aggregation through the intermediate function.<br />
</p><br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
[[image:HokkaidoU2012 Ag43 Figure4.jpg|left|thumb|400px|Fig. 4 Structure of Ag43 domain. This image is referenced from [1]. 5 motifs: RGD (orange), aspartyl protease (green), leucine zipper (pink), P-loop (blue) and another RGD (purple) exist in Ag43 complex.]]<br />
<p><br />
Signal peptide is present at the N-terminus of this protein and translated with &alpha;43 and &beta;43. In the presence of this signal, polypeptides consisted of other domains can cross from inner membrane to outer membrane. &beta;43 is mainly composed of &beta;-barrel structure. Some of membrane protein, particularly auto-transporter family has this domain which inserts into the outer membrane to work as anchor for passenger domain, and as pathway that allows passenger domain to cross from periplasm to outer membrane. Another domain which exists in the &beta;43, the auto-chaperon domain stabilizes in near the outer pore of &beta;-barrel then the domain associates folding of secreted passenger domain. &alpha;43 has &beta;-helical structure and some identified motifs, e.g., aspartyl protease site, a leucine zipper motif and RGD motif. But none of the motifs are conserved in all agn43 alleles.(Fig. 4) That means the motifs does not play a functional role but plays a role in the structural integrity [2]. <br />
</p><br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
[[image:HokkaidoU2012 Ag43 Figure5.JPG|right|thumb|300px|Fig. 5 Image of secretion of Ag43. IM is Innner Membrane, OM is Outer Membrane, SP is Signal Peptides, &alpha; is &alpha;43 domain and &beta; is &beta;43 domain. &alpha;43 forms &beta;-helix structure and &beta;43 forms &beta;-barrel structure.]]<br />
<p><br />
The actual secretional function of Ag43 is not still known. But about AIDA-I, One of the auto-transporter protein belonging to same group as Ag43, it is said that the AIDA-I passenger domain is auto-catalytically cleaved from complex with &beta;-barrel and autochaperon after secreted, then remain on the outside of cell membrane with non-covalently bound to translocator &beta;-barrel structure. This process would correspond to Ag43 because Ag43 is belonging to same family and has sequence similarity with AIDA-I [3]. It is generally confirmed that the structural and sequential similarity is correlated with functional similarity in proteomics. And the fact that the passenger domain is cleaved in the 60C will be additional evidence of non-covalent binding between each cell’s Ag43 [4]. The presence of transported Ag43 &alpha; domain is predicted in about 10nm of cell surface, so the cells that will aggregate with each other needs to be very close. If there were some obstacles near cells, they cannot aggregate. In other words, if there were rich other large proteins or cell surface structure, e.g., type 1 fimbriae and flagella on the cell membrane, Ag43 would not promote aggregation (Fig. 5) [5].<br />
</p><br />
</div><br />
<br />
==Experiments==<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 Ag43 Figure6 .JPG|left|thumb|380px|Fig. 6 Schematics of pBAD-RBS-Ag43-dT and pBAD-RBS-eCFP-RBS-Ag43-dT construct]]<br />
<p><br />
We appreciate to previous iGEM teams, especially iGEM 2010 Peking University team. We could easily get characterized Ag43 gene derived from K-12 strain flu gene. Compared with aggregation module of PKU iGEM 2010, our aggregation module is induced directly and executes aggregation more rapidly. We assembled a construct which is composed of pBAD+AraC (BBa_I0050) as promoter, ribosome binding site: RBS (BBa_B0034), Ag43 as protein coding region (BBa_K346007) and double terminator: dT (BBa_B0015) on a low copy plasmid pSTV28. (Fig. 6)<br />
</p><br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
[[image:HokkaidoU2012 Ag43 Figure7.JPG|right|thumb|300px|Fig. 7 An image of Ag43 expression induced by L-arabinose. If you added L-arabinose to cell culture, AraC binds with it and reveal from pBAD region and the construct is started to transcription.]] <br />
<p><br />
The induction of pBAD promoter is negatively controlled by AraC repressor. In the absence of L-arabinose, an AraC protein remains in pBAD promoter region and prevents transcription. In the presence of L-arabinose, AraC is released from pBAD by conformational change, and then RNA polymerase is allowed to start transcription of downstream sequence. Consequently, in this devise, we can induce Ag43 protein by addition of L-arabinose. And we inserted additional gene, eCFP: Cyan Fluorescent protein (BBa_E0020), upstream of Ag43 gene as indicator of expression of “Aggregation Module” (Fig. 7).<br />
</p><br />
<p><br />
Furthermore, we executed codon optimization of Ag43 by JCat [7] and replaced 6 recognition sites of PstI which originally exists in wild-type Ag43 sequence with sequences suitable for BioBrick assembly. By this optimization, we would be able to improve the translational frequency of Ag43 and accelerate the time required for aggregation.<br />
</p><br />
<br />
<br />
<p><br />
To aggregate cells each other, L-arabinose was added into LB liquid medium with appropriate antibiotics for induction of the function of “Aggregation Module”, and cultured in glass test tubes. The plastic tube was not suitable for producing large cluster of E. coli cells, since a strong affinity of E. coli expressing Ag 43 was observed onto the surface of inside of plastic test tube as shown in image 8. <br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image8 .JPG|center|thumb|400px|Image 8 Picture of cultivation in plastic tubes for 12 hrs and 16 hrs. Plasmid structure is pBAD-RBS-Ag43-dT-pSB1C3]]<br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
<p><br />
Meanwhile, we tested the same observation using 300 ml Erlenmeyer flask containing 50 ml LB liquid medium and cultivated at 34C (See [https://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_protocols protocol page] for experimental procedure).<br />
</p><br />
<p><br />
We found that optimum temperature required for efficient aggregation by “Aggregation Module” is 34~37C. We didn’t observe aggregation at 30C even after 24 hr-culture. <br />
Reciprocal shaking of culture flask seemed to be better than rotation.<br />
</p><br />
<p><br />
After culturing the aggregated E. coli, the clusters were harvested on nylon mesh by continuous strokes of injector (Fig. 9)(Image10). <br />
</p><br />
[[image:HokkaidoU2012 Ag43 Figure9.JPG|left|thumb|330px|Fig. 9 Image of harvesting method using nylon mesh by continuous strokes of injector. Only large clusters are harvested on nylon mesh]]<br />
[[image:HokkaidoU2012 Ag43 Image10.jpg|right|thumb|290px|Image10 Picture of our actual harvesting tools]]<br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
<p><br />
We succeeded in filtering them by nylon mesh which has 250 um pore size then we have gained E. coli cake (Image11). And as our experimental fact, we filtered them by 200 um pore size mesh too. This result shows that we would be able to gain target product by this devise if we cultivated E. coli which has ability to produce target product.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image11.JPG|center|thumb|350px|Image11 Cell cake on a nylon mesh (pore size: 250 um)]]<br />
<p><br />
Induction of function of “Aggregation Module” was tested by observing appearance of aggregated E. coli cluster in the presence (right) or absence (left) of L-arabinose in culture medium (See Image12). Aggregated E. coli clusters were observed only in the culture medium containing L-arabinose. The results suggest that the auto-aggregation of E. coli is mediated by overexpressed Ag43.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image12.jpg|center|thumb|300px|Image12 Induction of function of “Aggregation Module”. Appearance of aggregated E. coli in the presence (right) or absence (left) of L-arabinose in culture medium. Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 (low copy plasmid), and culture condition is at 37C for 24 hrs at 130 rpm.]]<br />
<p><br />
Besides above experiments, we tried to confirm a relationship between Ag43 expression and aggregation by monitoring signal associated with eCFP (indicator module) fused to “Aggregation Module”. During culture of the cells, we monitored change of OD600 as indicator of cell growth, and measured emission associated with eCFP as indicator of expression of “Aggregation Module” (See [https://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_protocols protocol page] for experimental procedure). Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3 and we cultivated at 37C for 12 hrs at 180 rpm.<br />
</p><br />
<p><br />
The result showed there is relation between optical density and aggregation. At the same time cultivating cells, OD600 of each cell culture's suspension are increasing, but increasing ratio of arabinose added culture (Arabinose+) became lower than negative control (Arabinose-) (Image13). On the other hand, we couldn't observe the expression of eCFP.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image13.JPG|center|thumb|400px|Image13 Graph of OD600 of suspension and culture time (hours). ]]<br />
</div><br />
<br />
==References==<br />
<div class="hokkaidou-section"><br />
[1] van der Woude, Marjan W Henderson, Ian R. Regulation and function of Ag43 (flu). Annu. Rev. Microbiol. 2008. 62:153–69 http://www.ncbi.nlm.nih.gov/pubmed/18785838<br />
<br />
<br />
[2] P Caffrey and P Owen. Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli. J. Bacteriol. 1989, 171(7):3634. http://www.ncbi.nlm.nih.gov/pubmed/2661530<br />
<br />
<br />
[3] Inga Benz, M. Alexander Schmidt. Structures and functions of autotransporter proteins in microbial pathogens. International Journal of Medical Microbiology 301 (2011) 461– 468. http://www.ncbi.nlm.nih.gov/pubmed/21616712<br />
<br />
<br />
[4]Glen C. Ulett, Jaione Valle, Christophe Beloin, Orla Sherlock, Jean-Marc Ghigo and Mark A. Schembri. Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract Infect. Immun. 2007, 75(7):3233. DOI: 10.1128/IAI.01952-06. http://iai.asm.org/content/75/7/3233.abstract<br />
<br />
<br />
[5] https://2010.igem.org/Team:Peking/Project/Bioabsorbent/InductiveAggregation<br />
<br />
<br />
[6] http://openwetware.org/wiki/Arking:JCAOligoTutorial8<br />
<br />
<br />
[7] http://www.jcat.de/<br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Aggregation
Team:HokkaidoU Japan/Project/Aggregation
2012-09-27T03:32:25Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
==Planning of "Aggregation Module"==<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 Ag43 Figure1.JPG|right|thumb|300px|Fig. 1 Large amount of cultivated cells is time-consuming to harvest by centrifugation]]<br />
<p><br />
In modern industry, two major methods to harvest cultured cells that produce high-value added macromolecules are centrifugation and filtration. Since sizes of Escherichia coli cells are very small, harvesting them by filtration is difficult. So the most popular method for harvesting E. coli is centrifugation. However centrifugation is time-consuming process particularly when harvest them from large volume of culture medium. (Fig. 1)<br />
</p><br />
<p><br />
The purpose of this project is to make more efficient method for macromolecule production in bacterial cells by eliminating time-consuming centrifugation step, through enlarging sizes of E. coli cell clusters (large enough to be captured), named “Bio-capsule”, for harvesting them by filtration on nylon mesh.<br />
</p><br />
<p><br />
For this purpose, we made E. coli expressing adhesion molecule, Antigen 43 (Ag43), on the surface of outer membrane of it for enhancing cell-cell interaction. We have succeeded in harvesting large amount of E. coli cells as filter cake on nylon mesh after continuous rapid strokes of injector (Fig. 2). The “Bio-capsule” resulted from function of the “Aggregation Module” will contribute to the industrial production in near future.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Figure2.JPG|center|thumb|400px|Fig. 2 Our capturing tools and harvesting method]]<br />
</div><br />
<br />
==Background of "Aggregation Module"==<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 Ag43 Figure3 .JPG|center|thumb|500px|Fig. 3 Cell-to-cell aggregation by Ag43 complex. &alpha;43 domain exists at 10 nm above on the E. coli cell surface by non-covelent bond with &beta;43 domain (right). Left picture is actual cluster observed with optical microscopy.]]<br />
<p><br />
Ag43 is one of a kind of outer membrane proteins and belongs to auto-transporter family. This protein was derived from E. coli locus: flu, mapping on the E. coli K-12 chromosome. Other E. coli strains also have same gene, but this K-12 strain’s Ag43 cause the most rapid aggregation compared with other strain’s Ag43 [2]. Typical auto-transporter domains: signal peptides, N-proximal passenger domain (&alpha;43 : processing sites), autochaperon domain and C-terminal β-barrel domain (&beta;43 : translocation unit) are found in the protein. These domains promote cell-to-cell auto-aggregation through the intermediate function.<br />
</p><br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
[[image:HokkaidoU2012 Ag43 Figure4.jpg|left|thumb|400px|Fig. 4 Structure of Ag43 domain. This image is referenced from [1]. 5 motifs: RGD (orange), aspartyl protease (green), leucine zipper (pink), P-loop (blue) and another RGD (purple) exist in Ag43 complex.]]<br />
<p><br />
Signal peptide is present at the N-terminus of this protein and translated with &alpha;43 and &beta;43. In the presence of this signal, polypeptides consisted of other domains can cross from inner membrane to outer membrane. &beta;43 is mainly composed of &beta;-barrel structure. Some of membrane protein, particularly auto-transporter family has this domain which inserts into the outer membrane to work as anchor for passenger domain, and as pathway that allows passenger domain to cross from periplasm to outer membrane. Another domain which exists in the &beta;43, the auto-chaperon domain stabilizes in near the outer pore of &beta;-barrel then the domain associates folding of secreted passenger domain. &alpha;43 has &beta;-helical structure and some identified motifs, e.g., aspartyl protease site, a leucine zipper motif and RGD motif. But none of the motifs are conserved in all agn43 alleles.(Fig. 4) That means the motifs does not play a functional role but plays a role in the structural integrity [2]. <br />
</p><br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
[[image:HokkaidoU2012 Ag43 Figure5.JPG|right|thumb|300px|Fig. 5 Image of secretion of Ag43. IM is Innner Membrane, OM is Outer Membrane, SP is Signal Peptides, &alpha; is &alpha;43 domain and &beta; is &beta;43 domain. &alpha;43 forms &beta;-helix structure and &beta;43 forms &beta;-barrel structure.]]<br />
<p><br />
The actual secretional function of Ag43 is not still known. But about AIDA-I, One of the auto-transporter protein belonging to same group as Ag43, it is said that the AIDA-I passenger domain is auto-catalytically cleaved from complex with &beta;-barrel and autochaperon after secreted, then remain on the outside of cell membrane with non-covalently bound to translocator &beta;-barrel structure. This process would correspond to Ag43 because Ag43 is belonging to same family and has sequence similarity with AIDA-I [3]. It is generally confirmed that the structural and sequential similarity is correlated with functional similarity in proteomics. And the fact that the passenger domain is cleaved in the 60C will be additional evidence of non-covalent binding between each cell’s Ag43 [4]. The presence of transported Ag43 &alpha; domain is predicted in about 10nm of cell surface, so the cells that will aggregate with each other needs to be very close. If there were some obstacles near cells, they cannot aggregate. In other words, if there were rich other large proteins or cell surface structure, e.g., type 1 fimbriae and flagella on the cell membrane, Ag43 would not promote aggregation (Fig. 5) [5].<br />
</p><br />
</div><br />
<br />
==Experiments==<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 Ag43 Figure6 .JPG|left|thumb|380px|Fig. 6 Schematics of pBAD-RBS-Ag43-dT and pBAD-RBS-eCFP-RBS-Ag43-dT construct]]<br />
<p><br />
We appreciate to previous iGEM teams, especially iGEM 2010 Peking University team. We could easily get characterized Ag43 gene derived from K-12 strain flu gene. Compared with aggregation module of PKU iGEM 2010, our aggregation module is induced directly and executes aggregation more rapidly. We assembled a construct which is composed of pBAD+AraC (BBa_I0050) as promoter, ribosome binding site: RBS (BBa_B0034), Ag43 as protein coding region (BBa_K346007) and double terminator: dT (BBa_B0015) on a low copy plasmid pSTV28. (Fig. 6)<br />
</p><br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
[[image:HokkaidoU2012 Ag43 Figure7.JPG|right|thumb|300px|Figure7 An image of Ag43 expression induced by L-arabinose. If you added L-arabinose to cell culture, AraC binds with it and reveal from pBAD region and the construct is started to transcription.]] <br />
<p><br />
The induction of pBAD promoter is negatively controlled by AraC repressor. In the absence of L-arabinose, an AraC protein remains in pBAD promoter region and prevents transcription. In the presence of L-arabinose, AraC is released from pBAD by conformational change, and then RNA polymerase is allowed to start transcription of downstream sequence. Consequently, in this devise, we can induce Ag43 protein by addition of L-arabinose. And we inserted additional gene, eCFP: Cyan Fluorescent protein (BBa_E0020), upstream of Ag43 gene as indicator of expression of “Aggregation Module” (Fig. 7).<br />
</p><br />
<p><br />
Furthermore, we executed codon optimization of Ag43 by JCat [7] and replaced 6 recognition sites of PstI which originally exists in wild-type Ag43 sequence with sequences suitable for BioBrick assembly. By this optimization, we would be able to improve the translational frequency of Ag43 and accelerate the time required for aggregation.<br />
</p><br />
<br />
<br />
<p><br />
To aggregate cells each other, L-arabinose was added into LB liquid medium with appropriate antibiotics for induction of the function of “Aggregation Module”, and cultured in glass test tubes. The plastic tube was not suitable for producing large cluster of E. coli cells, since a strong affinity of E. coli expressing Ag 43 was observed onto the surface of inside of plastic test tube as shown in image 8. <br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image8 .JPG|center|thumb|400px|Image 8 Picture of cultivation in plastic tubes for 12 hrs and 16 hrs. Plasmid structure is pBAD-RBS-Ag43-dT-pSB1C3]]<br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
<p><br />
Meanwhile, we tested the same observation using 300 ml Erlenmeyer flask containing 50 ml LB liquid medium and cultivated at 34C (See [https://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_protocols protocol page] for experimental procedure).<br />
</p><br />
<p><br />
We found that optimum temperature required for efficient aggregation by “Aggregation Module” is 34~37C. We didn’t observe aggregation at 30C even after 24 hr-culture. <br />
Reciprocal shaking of culture flask seemed to be better than rotation.<br />
</p><br />
<p><br />
After culturing the aggregated E. coli, the clusters were harvested on nylon mesh by continuous strokes of injector (Fig. 9)(Image10). <br />
</p><br />
[[image:HokkaidoU2012 Ag43 Figure9.JPG|left|thumb|330px|Fig. 9 Image of harvesting method using nylon mesh by continuous strokes of injector. Only large clusters are harvested on nylon mesh]]<br />
[[image:HokkaidoU2012 Ag43 Image10.jpg|right|thumb|290px|Image10 Picture of our actual harvesting tools]]<br />
<div style="clear: both; width: 100%; height: 20px;"></div><br />
<p><br />
We succeeded in filtering them by nylon mesh which has 250 um pore size then we have gained E. coli cake (Image11). And as our experimental fact, we filtered them by 200 um pore size mesh too. This result shows that we would be able to gain target product by this devise if we cultivated E. coli which has ability to produce target product.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image11.JPG|center|thumb|350px|Image11 Cell cake on a nylon mesh (pore size: 250 um)]]<br />
<p><br />
Induction of function of “Aggregation Module” was tested by observing appearance of aggregated E. coli cluster in the presence (right) or absence (left) of L-arabinose in culture medium (See Image12). Aggregated E. coli clusters were observed only in the culture medium containing L-arabinose. The results suggest that the auto-aggregation of E. coli is mediated by overexpressed Ag43.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image12.jpg|center|thumb|300px|Image12 Induction of function of “Aggregation Module”. Appearance of aggregated E. coli in the presence (right) or absence (left) of L-arabinose in culture medium. Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 (low copy plasmid), and culture condition is at 37C for 24 hrs at 130 rpm.]]<br />
<p><br />
Besides above experiments, we tried to confirm a relationship between Ag43 expression and aggregation by monitoring signal associated with eCFP (indicator module) fused to “Aggregation Module”. During culture of the cells, we monitored change of OD600 as indicator of cell growth, and measured emission associated with eCFP as indicator of expression of “Aggregation Module” (See [https://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_protocols protocol page] for experimental procedure). Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3 and we cultivated at 37C for 12 hrs at 180 rpm.<br />
</p><br />
<p><br />
The result showed there is relation between optical density and aggregation. At the same time cultivating cells, OD600 of each cell culture's suspension are increasing, but increasing ratio of arabinose added culture (Arabinose+) became lower than negative control (Arabinose-) (Image13). On the other hand, we couldn't observe the expression of eCFP.<br />
</p><br />
[[image:HokkaidoU2012 Ag43 Image13.JPG|center|thumb|400px|Image13 Graph of OD600 of suspension and culture time (hours). ]]<br />
</div><br />
<br />
==References==<br />
<div class="hokkaidou-section"><br />
[1] van der Woude, Marjan W Henderson, Ian R. Regulation and function of Ag43 (flu). Annu. Rev. Microbiol. 2008. 62:153–69 http://www.ncbi.nlm.nih.gov/pubmed/18785838<br />
<br />
<br />
[2] P Caffrey and P Owen. Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli. J. Bacteriol. 1989, 171(7):3634. http://www.ncbi.nlm.nih.gov/pubmed/2661530<br />
<br />
<br />
[3] Inga Benz, M. Alexander Schmidt. Structures and functions of autotransporter proteins in microbial pathogens. International Journal of Medical Microbiology 301 (2011) 461– 468. http://www.ncbi.nlm.nih.gov/pubmed/21616712<br />
<br />
<br />
[4]Glen C. Ulett, Jaione Valle, Christophe Beloin, Orla Sherlock, Jean-Marc Ghigo and Mark A. Schembri. Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract Infect. Immun. 2007, 75(7):3233. DOI: 10.1128/IAI.01952-06. http://iai.asm.org/content/75/7/3233.abstract<br />
<br />
<br />
[5] https://2010.igem.org/Team:Peking/Project/Bioabsorbent/InductiveAggregation<br />
<br />
<br />
[6] http://openwetware.org/wiki/Arking:JCAOligoTutorial8<br />
<br />
<br />
[7] http://www.jcat.de/<br />
</div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/File:HokkaidoU2012_Nile-red2.png
File:HokkaidoU2012 Nile-red2.png
2012-09-27T03:25:33Z
<p>Slecat: </p>
<hr />
<div></div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:10:35Z
<p>Slecat: /* Staining by Nile red */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red an P3HB was detected only in the presence of plastic producing module(right panel in figure5), but not in the absence of it(left panel in figure 4)<br />
<br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed <br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/File:HokkaidoU2012_Nilered.jpg
File:HokkaidoU2012 Nilered.jpg
2012-09-27T03:09:54Z
<p>Slecat: </p>
<hr />
<div></div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:02:04Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
<br />
<br />
[[image:HokkaidoU2012_Nilered-pika.jpg|center|thumb|600px|hogehoge2]]<br />
[[image:HokkaidoU2012 Nilered-non.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed <br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/File:HokkaidoU2012_Nilered-pika.jpg
File:HokkaidoU2012 Nilered-pika.jpg
2012-09-27T02:54:06Z
<p>Slecat: </p>
<hr />
<div></div>
Slecat
http://2012.igem.org/File:HokkaidoU2012_Nilered-non.jpg
File:HokkaidoU2012 Nilered-non.jpg
2012-09-27T02:53:35Z
<p>Slecat: </p>
<hr />
<div></div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_11
Team:HokkaidoU Japan/Notebook/plastic Week 11
2012-09-26T20:21:59Z
<p>Slecat: /* Digestion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 10th==<br />
<div><br />
==PCR==<br />
<p><br />
PhaA was multiplied from pGEM with XbaI and SpeI restriction sites by PCR.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
PhaA was digested with XbaI and SpeI. RBS on pSB1A2 was digested with SpeI.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
We confirmed succession of digestion by electrophoresis.<br/><br />
And then DNA were extracted from TBE gel.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
RBS on pSB1A2 was ligated with PhaA and PhaB.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
These ligated DNAs transformed into E.coli (strain: DH5&alpha;).<br/><br />
And then we spread fungus liquid added LB on plates.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 11th==<br />
<div><br />
==Sequencing==<br />
<p><br />
We analyzed the sequence which contains phaA to check whether there are mutations.<br /><br />
<br />
<br />
</p><br />
<br />
==Colony PCR==<br />
<br />
The colony of RBS+phaB on 1A2 was <br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 12th==<br />
<div><br />
==Digestion==<br />
<p><br />
RBS-PhaB on pSB1A2 was digested with EcoRI and SpeI restriction sites and dT(B0015) on pSB1AK3 was also digested with EcoRI and XbaI.<br />
<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
The fragment of RBS-PhaB and dT(B0015) on pSB1AK3 was ligated.<br /><br />
However, because of my stupid mistake, both fragment failed to ligate.<br />
I deeply feel sorry to Mr.Kawata and the E. coli.<br />
<br />
<br />
</p><br />
<br />
==Polymer extraction==<br />
<br />
<p><br />
@Taguchi lab<br /><br />
The dried cells were removed to glass test tubes.<br />
Weight of plastic tubes were measured in order to estimate how much the cells weighed.<br />
<br />
# pantothenic acid (+) 1.5 ml W:1.466 (W=tube weight with dried cells inside.)<br />
# pantothenic acid (-) 1.5 ml W:1.467<br />
# pantothenic acid (+) 0.7 ml W:1.461<br />
<br />
DH5a<br /><br />
# pantothenic acid (+) 1.5 ml W:1.468<br />
# pantothenic acid (-) 1.5 ml W:1.468<br />
# pantothenic acid (+) 0.7 ml W:1.459<br />
<br />
We couldn't remove all the cells to the glass tubes because the cells remained on the plastic tubes walls.<br />
The weight of plastic tubes were measured again to estimate the amount of removed cells.<br />
<br />
# pantothenic acid (+) 1.5 ml W:1.461 (W=tube weight with nothing inside.)<br />
# pantothenic acid (-) 1.5 ml W:1.467<br />
# pantothenic acid (+) 0.7 ml W:1.458<br />
<br />
DH5a<br /><br />
# pantothenic acid (+) 1.5 ml W:1.462 <br />
# pantothenic acid (-) 1.5 ml W:1.464<br />
# pantothenic acid (+) 0.7 ml W:1.459<br />
<br />
From these data, we can estimate the weight of the dried cells.<br />
<br />
# pantothenic acid (+) 1.5 ml W:0.144 (W=weight of dried cells.)<br />
# pantothenic acid (-) 1.5 ml W:0.099<br />
# pantothenic acid (+) 0.7 ml W:0.039<br />
<br />
DH5a<br /><br />
# pantothenic acid (+) 1.5 ml W:0.007<br />
# pantothenic acid (-) 1.5 ml W:0.006<br />
# pantothenic acid (+) 0.7 ml W:-----<br />
<br />
<br />
<br />
<br />
<br />
Chloroform was added to the dried cells and was incubated on 60C for 48 hrs.<br />
<br />
<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 13th==<br />
<div><br />
==Digestion==<br />
<p><br />
We were worried about yesterdays lab work, so we tried it again.<br/><br />
RBS-PhaB on pSB1A2 was digested with EcoRI and SpeI restriction sites and dT(B0015) on pSB1AK3 was also digested by EcoRI and XbaI.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
We confirmed the succession of digestion by electrophoresis, and extracted the DNAs from TBE gel.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
The DNA solution of RBS-PhaB and dT on pSB1AK3 were concentrated by EtOH precipitation.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
RBS-PhaB was ligated with dT on pSB1AK3.<br/><br />
We added LB to fungus liquid mixed ligated DNAs and then spread it on LBK plates.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 14th==<br />
<div><br />
<br />
==Digestion==<br />
<p><br />
To submit our construct as a BioBrick, we had to ligate our part samples in pSB1C3.<br /><br />
RBS-phaC on pSB1A2, RBS-phaA and pSB1C3 was digested by EcoRI and PstI.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
We confirmed the succession of digestion by electrophoresis, and extracted the DNAs from TBE gel.<br />
</p><br />
<br />
==Polymer extraction==<br />
<p><br />
The samples were filiterized and were removed to another test tubes.<br />
I made a 1.7 mL loss of sample2.<br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
We confirmed whether or not RBS-PhaB was ligated with dT on pSB1AK3 correctly by colony PCR.<br/><br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
We added 2 ml LB with 2 ul kanamycin to pre-cultured colony suspension, and then started to incubate at 37C, 180 rpm.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 15th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmids of RBS-PhaB-dT on pSB1AK3 were extracted.<br/><br />
And then we got DNA solution of it.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
RBS-PhaB-dT on pSB1AK3 was digested with XbaI and PstI restriction sites.<br/><br />
RBS-PhaC-RBS-PhaA on pSB1A2 was also digested by SpeI and PstI.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
We confirmed succession of digestion by electrophoresis.<br/><br />
Digested DNAs were extracted from TBE gel.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
The digested DNA were condensed by EtOH precipitation.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
RBS-PhaB-dT was ligated with RBS-PhaC-RBS-PhaA on pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
The ligated DNA was transformed into E. coli (strain: JM109).<br/><br />
E. coli solution was spread on LB.<br />
</p><br />
<br />
---------<br />
<br />
==Gel extraction==<br />
<p><br />
The plasmids of [RBS-phaB on pSB1C3] and [RBS-phaC-RBS-phaA on pSB1C3] were extracted by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
[RBS-phaA] and [RBS-phaC] were condensed by Ethanol precipitation.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
[RBS-phaA] and [RBS-phaC] was ligated with pSB1C3.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
[RBS-phaB on pSB1A2] was digested by EcoRI and PstI.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 16th==<br />
<div><br />
<br />
==Colony PCR==<br />
<p><br />
We confirmed the ligation of RBS-PhaB-dT and RBS-PhaC-RBS-PhaA on pSB1A2 by colony PCR.<br/><br />
</p><br />
<br />
<br />
==Liquid culture==<br />
<p><br />
We added 2 ml LB and 2 ul antibiotic to pre-cultured colony suspension.<br/><br />
And then started to incubate at 37C, 180 rpm.<br/><br />
Bacteria hold BBa_J23109, BBa_J23114, BBa_J23101 and BBa_J23102, constitutive promoter each of them have different expression intensity and pTet(BBa_R0040) were also begun to incubate.<br />
</p><br />
<br />
---------<br />
<br />
==Colony PCR==<br />
<p><br />
We confirmed the ligation of [RBS-phaA on pSB1C3] and [RBS-phaC on pSB1C3] by colony PCR.<br/><br />
</p><br />
<br />
==Ligation==<br />
<br />
<p><br />
[RBS-phaB] was ligated with pSB1C3.<br />
</p><br />
<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_1
Team:HokkaidoU Japan/Notebook/aggregation Week 1
2012-09-26T20:11:27Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 2nd==<br />
<div><br />
On your mark...<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 3rd==<br />
<div><br />
Get set...<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 4th==<br />
<div><br />
===Transformation===<br />
<p><br />
Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) into DH5&alpha;<br />
#Mixed 1 ul DNA solution with DH5&alpha; competent cells and incubated on ice for 30 min.<br />
#Plated them on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). ]<br />
To gain chloramphenicol resistances transformed cell solution was incubated for 2 hrs.<br />
pT7 plate was incubated for 21 hrs and Others for 20 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 5th==<br />
<div><br />
===Transformation===<br />
<p><br />
'''K346007(Ag43) failed to grow on LBC plate.'''<br />
<br />
<br />
Transformation of K346007(Ag43) into DH5&alpha;.<br />
#Mixed 1 ul DNA solution with DH5&alpha; competent cells and incubated on ice for 30 min.<br />
#To gain chrolamphenicol resistance solution was pre-incubated for 2 hrs.<br />
#Incubated on LBC for 21 hrs.<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Performed single colony isolation of BBa_B0015, B0034, I179005 and K542009.<br />
#Incubated the plates for 14 hrs and 30 mins <br />
<br />
After consulting part page we found out that<br />
'''BBa_K542009 was Ag43 basic part, not composite! And the part didn't have Biobrick suffix.'''<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 6th==<br />
<div><br />
===Incubation for plasmid extraction and glycerol stock===<br />
<p><br />
Incubated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) solution.<br />
#Picked up two colonies from each plate.<br />
#First colony was re-suspended in 1 ml LB (A and C respectively) for glycerol stock. Second colony was re-suspended in 2 ml LB(A and C respectively) for plasmid extraction. <br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
#Single colony isolation of K346007(Ag43) on LBC. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 7th==<br />
<div><br />
===Incubation of plasmid extraction and glycerol stock===<br />
<p><br />
Incubation of (Ag43) in LBC liquid medium.<br />
<br />Two colonies were re-suspended in 2 ml LBC. We incubated them at 38C.<br /><br />
<br />
'''One of the tubes was incubated for 8 hrs. It's for glycerol stock.'''<br />
</p><br />
<br />
===3A assembly!!!===<br />
<p><br />
Assembled pT7, RBS and pSB1C3 by 3A assembly.<br />
This is our first try!<br />
</p><br />
<br />
===Plasmid extraction===<br />
<p><br />
#Plasmid extraction of dT,RBS,pT7 and pLacI-RBS-Ag43. We used FastGene Plasmid Mini Kit(Nippon Gene)<br />
#We got 50 ul of DNA solutions.<br />
</p><br />
<br />
===Glycerol stock===<br />
<p><br />
Glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.<br />
#Parts written above were incubated in 1 ml of LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) for 16 hrs 30 min.<br />
#Add glycerol and Freeze at -80C<br />
</p><br />
<br />
===Estimation of plasmid concentration===<br />
<p><br />
[[Image:120707_I719005_B0034_B0015_K542009_mini-prep_umeuchi.jpg|thumb|Erectrophoresis result]]<br />
Electrophoresis to estimate the concentration of plasmid extraction products(dT,RBS,pT7 and pLacI-RBS-Ag43).<br />
#Used 1% agarose gel.<br />
#Added EtBr to TBE buffer and soaked the gel by applying current.<br />
#Ran '''1.2 ul of DNA solutions (1 ul is plasmid extraction product and 0.2 ul is Loading Dye)''' for 35 min.<br />
#Took a photograph of 1% agarose gel.<br />
</p><br />
<br />
===Digestion of I719005, B0034 and pSB1K3===<br />
<p><br />
<br />
<br />
Digestion reaction<br />
<br />
All parts were digested in 30 ul reaction solution.<br />
*I719005(40 ng/ul)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|12.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH Buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|}<br />
<br />
<br />
*B0034(40 ng/ul)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM Buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|}<br />
<br />
<br />
*pSB1K3(25 ng/ul)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH Buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation of Digestion products===<br />
<p><br />
Concentrating the DNA solution which and removing restriction enzymes.<br />
#Added 3 ul of NaoAc, 1.5 ul of glycogen and 75 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air dried at room temperature. Dissolved in 10 ul of DW.<br />
</p><br />
<br />
===Ligation (3A Assembly)===<br />
<p><br />
3A assembly requires Ligation reaction total volume to be 25 ul.<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ligation Mighty Mix<br />
|12.5 ul<br />
|-<br />
|pT7<br />
|2 ul<br />
|-<br />
|RBS<br />
|2 ul<br />
|-<br />
|pSB1K3<br />
|2 ul<br />
|-<br />
|DW<br />
|6.5 ul<br />
|-<br />
|Total<br />
|25 ul<br />
|}<br />
<br />
Incubation of ligation reaction.<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br /><br />
<br />
ligation was finished. <br />
But now is 10 pm. 2.5hrs are needed for transformation. Transformation would be finished at 0:30 am.<br />
<br />
Withdraw!!!!<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 8th==<br />
<div><br />
*(pT7 + RBS)<br />
===Transformation===<br />
<p><br />
Transformation for pT7+RBS+pSB1K3<br />
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Stood on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubated for 2 hrs at 37C.<br />
#Splead 300 ul of LB&DH5&alpha; solution to LBK.<br />
#Incubated for 19 hrs.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for Liquid culture product of K346007(Ag43)<br />
#Used FastGene Plasmid Mini Kit(Nippon Genetics)<br />
#Elutioned in 50 ul<br />
#'''First we eluted in collection tube. then moved in micro-centrifuge tube.''' <br />
</p><br />
===Electrophoresis===<br />
<p><br />
[[image:HokkaidoU2012 120708 K346007 miniprep and pt7 rbs.jpg|thumb|plasmid extraction result (With ligation result of pT7+RBS+pSB1K3)]]<br />
Electrophoresis for plasmid extraction product(Ag43).<br />
#Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.<br />
#1 ul 1kb ladder, 1.2 ul plasmid extraction product(1 ul is DNA solution and 0.2 ul is loading dye) added then migtrated.<br />
</p><br />
===Glycerol stock===<br />
<p><br />
Made glycerol stock of K346007 (Ag43).<br />
#Parts written above were incubated in LBC.<br />
#Added glycerol and freezed at -80C<br />
</p><br />
*(Ag43 + dT)<br />
Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)<br />
===Digestion===<br />
<p><br />
Digested Ag43 and dT in solution by recipes Written below.<br />
'''Insert DNA required too much weight and volume'''(volume was calculated from concentration of plasmid extraction<br />
product)from our calculation. There are no insurance of succession of digestion. <br />
*Ag43(Insert)<br />
5190bp(Ag43 + pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|48 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|6 ul<br />
|-<br />
DW<br />
|4 ul<br />
|-<br />
|Total<br />
|60 ul<br />
|}<br />
*dT(Vector)<br />
3318bp(Ag43 + pSB1AK3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
[[image:HokkaidoU2012 120708 dt K346007 digestion umeuchi.jpg|thumb|Digestion result]]<br />
K346007(Ag43) was 5190bp before digestion (Biobrick prefix + Ag43 + Biobrick suffix + pSB1C3). <br />
After digestion, Ag43 and pSB1C3 was separated and became fragments about 3120bp(Ag43) and 2070bp(pSB1C3).<br />
Gel image above shows there are two fragments, one fragment is about 2000bp other fragment is 3000bp.<br />
Digestion would be succeeded.<br />
About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.<br />
</p><br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))<br />
#Added 5 ul of NaoAc , 1.5 ul of glycogen and 125 ul of 100% ethanol to 50 ul DNA solutions.<br />
#Centrifuged at 15000 rpm, 10min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW.<br />
</p><br />
===Ligation===<br />
<p><br />
All DNA solutions were digested.<br />
Used Ligation Mighty Mix(TakaraBio)<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Insert: Ag43<br />
|2 ul<br />
|-<br />
|Vector: dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
Ligation reaction recipe is following.<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
===Electrophoresis===<br />
<p><br />
[[image:HokkaidoU2012 120708 K346007-dT ligation.jpg|thumb|Erectrophoresis result]]<br />
Confirmation of succession of ligation.<br />
#Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.<br />
#Added 1kb ladder, Ligation product(1 ul) and digestion products (control:each solutions 1 ul).<br />
#Migtrated for 30 min.<br />
</p><br />
===Transformation===<br />
<p><br />
Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.<br />
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#From 700 solution(100 ul is DH5&alpha; and 600 ul is LB), 100 ul add to 900 ul of LB(x10 solution)<br />
#Spread 300 ul from 600(700-100) ul and 1000 ul of LB&DH5&alpha; solution to each LBA plates.<br />
#Incubated for 19 hrs.<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_12
Team:HokkaidoU Japan/Notebook/aggregation Week 12
2012-09-26T20:10:40Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 17th==<br />
<div><br />
==Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2==<br />
<p><br />
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI.<br />
<br />
Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (45~50 ng/ul)<br />
|27 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|40 ul<br />
|}<br />
<br />
<br />
Vector(ptet-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (60~70 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (20~30 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 17 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 18th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==<br />
<p><br />
<br />
We did colony PCR two times.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(200bp down primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-f4 primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 702bp.<br />
<br />
[[image:HokkaidoU2012 120918 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120918 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result 2]]<br />
<br />
<br />
Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once more.<br />
</p><br />
<br />
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI and pSB1C3 with EcoRI and SpeI.<br />
<br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 35ng/ul)<br />
|41 ul<br />
|-<br />
|EcoRI<br />
|2 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xH buffer<br />
|5 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
Vector(pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg |thumb|digestion result]]<br />
<br />
</p><br />
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA <br />
|4 ul<br />
|-<br />
|Vector DNA<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
Transformation ligation product into JM109.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 19th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
<br />
Colony PCR to confirm whether pSTV28 vettor has pBAS-RBs-eCFP-RBs-Ag43-dT as insert or not.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(200bp down primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-f4 primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 702bp.<br />
<br />
[[image:HokkaidoU2012 120919 colop2 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]<br />
<br />
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.<br />
</p><br />
<br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3 and pBAD-RBS-eCFP-RBS-Ag43-dI on pSB1C3==<br />
<p><br />
<br />
Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can anneal to a part of eCFP coding site and another can anneal to a part of Ag43 coding site.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(FP-F primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-R primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1, N2 (DW only) as controls.<br />
Desired product is about 452 bp and 697 bp respectively.<br />
<br />
[[image:HokkaidoU2012 120919 colop3 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]<br />
<br />
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 20th==<br />
<div><br />
<p><br />
==Digestion of ptet-RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 (DNA solution eluted from colony No.10, 12 respectively in ) and ptet-pSB1A2==<br />
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI. We prepared DNa solution derived from No. 10 and No. 12 colony selected selected by the result of colony PCR for 18th.<br />
<br />
Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Insert No.12 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(ptet-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120919 digestion InsertNo10-InsertNo12-Vector.jpg|thumb|digestion result]]<br />
<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120919 Ethapre InsertNo10-InsertNo12-Vector.jpg|thumb|ethanol precipitation result]]<br />
We decided to use No. 10 digestion product for ligation, and confirmed that the concentration of Insert DNA solution is 50 ng/ul and Vector DNA solution is about 20~30 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (50 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (20~30 ng/ul)<br />
|1.5 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 21th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(X-phaB-F primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaC-RBs-phaA-RBS-phaB-pSB1A2)as controls.<br />
Desired product is about 1500bp.<br />
<br />
[[image:HokkaidoU2012 120921 colop1 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120921 colop2 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result2]]<br />
<br />
<br />
Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for plasmid extraction and No. 6, 12, 14 for storing at 4C. <br />
</p><br />
</div></div><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_11
Team:HokkaidoU Japan/Notebook/aggregation Week 11
2012-09-26T20:10:00Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 10th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 by EcoRI and SpeI, Ag43-dT-pSB1AK3 (previously digested by HindIII) by XbaI and NotI, and pSTV28 by EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 by XbaI and SpeI as a control for confirmation of the ability of restriction enzyme.<br />
<br /><br />
Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
Insert2 (Ag43-dT-pSB1AK3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|25 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xK buffer<br />
|1.5 ul<br />
|-<br />
|100xBSA<br />
|0.3ul<br />
|-<br />
|DW<br />
|1.5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (15 ng/ul)<br />
|9 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|10xBSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30~40 ng/ul)<br />
|10 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120910 digestion Insert1-pBAD-RBS-eCFP-RBS-pSB1A2 Insert2-Ag43-dT-pSB1AK3 Vector-ptet-RBS-eYFP-dT-pSTV28 Control-pT7-RBS-pSB1A2.jpg|thumb|digestion result]]<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120910 EthaPre Insert1-Insert2-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert1 DNA solution is 50 ng/ul, Insert2 DNA solution is 10~15 ng/ul and Vector DNA solution is 10 ng/ul.<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (10 ng/ul)<br />
|4 ul<br />
|-<br />
|Insert1 DNA (50 ng/ul)<br />
|2 ul<br />
|-<br />
|Insert2 DNA (10~15 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
Transformation ligation product into DH5&alpha;.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 14 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 11th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2==<br />
<p><br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
*We noticed that this step was actually 68.9 degree after reaction. <br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 542bp.<br />
<br />
[[image:HokkaidoU2012 120911 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
Because of mis-setting the PCR program, we failed to amplify desired DNA fragment so we retried the colony PCR in same reagent, correct PCR machine program.<br />
<br />
[[image:HokkaidoU2012 120912 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed that the ligated DNA contains at least Ag43-dT-BioBrick_Suffix complex. We selected No. 2,4 for incubation for plasmid extraction and No. 5,6 for Aggregation check.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 12th==<br />
<div><br />
==Analysis nucleotide sequence==<br />
<p><br />
We analyzed the nucleotide sequence of pBAD-RBS-Ag43-dT on pSB1AK3 and pSB1C3.<br />
We used these 6 kinds of primers.<br />
100b up EX-F primer, pBAD-f1 primer, pBAD-f2 primer, Ag43-f1 primer, Ag43-f2 primer, Ag43-f3 primer, Ag43-f4 primer, 200b down PS-R primer<br />
<br />
</p><br />
<br />
==Aggregation check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
Aggregation check for No. 5,6 colonies selected by colony PCR at August 11th.<br />
#Prepared 5 ml LBA into glass tubes.<br />
#Re-suspended 2 colony mixture (No. 2 and No. 5 respectively). <br />
#Incubated at 37C for 18 hrs.<br />
</p><br />
<br />
==Digestion of RBS-phaB-pSB1A2 as Vector and RBS-eYFP-dT as Insert==<br />
<p><br />
To make a construct of RBS-phaB-RBS-eYFP-dT-pSB1A2, we digested RBS-phaB-pSB1A2 with SpeI & PstI, RBS-eYFP-dT with XbaI & PstI.<br />
<br />
<br />
Insert (RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|25 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xK buffer<br />
|1.5 ul<br />
|-<br />
|100xBSA<br />
|0.3 ul<br />
|-<br />
|DW<br />
|1.5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS-phaB-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120912 Digestion Insert(RBS-eYFP-dT)-Vector(RBS-phaB-pSB1A2)-Control(pBAD-RBS-eCFP-RBS-pSB1A2).jpg|thumb|digestion result]]<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120912 Ethapre 20min Insert-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 40 ng/ul and Vector DNA solution is 30 ng/ul.<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (40 ng/ul)<br />
|3 ul<br />
|-<br />
|Insert DNA (30 ng/ul)<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 13th==<br />
<div><br />
==Transformation of RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 14 hours.<br />
</p><br />
<br />
==Aggregation Check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
#Mixed 25 ul Arabinose solution (20 %) to 5 ml LBC in glass tube.<br />
#Incubated for 24 hrs at 37C.<br />
<br />
<br />
Result: The construct has aggregated not forming large cluster but small dot cluster, and the supernatant has muddiness.<br />
</p><br />
<br />
==Colony PCR of RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R down primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|64.4<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaB-pSB1A2)as controls.<br />
Desired product is about 1800~2000bp.<br />
<br />
[[image:HokkaidoU2012 120913 colop RBS-phaB-RBS-eYFP-dT-pSB1A2 1,2,,,N1,N2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.<br />
</p><br />
<br />
<br />
==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 1 and No. 2 respectively). <br />
#Incubated at 37C for 14 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 14th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.<br />
<br />
Insert (RBS-phaB-RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|21 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(RBS-phaC-RBS-phaA-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120914 Digestion Insert Vector.jpg|thumb|digestion result]]<br />
<br />
We suppose the Insert DNA (RBS-phaB-RBS-eYFP-dT) has contained Vector-dimer DNA as contamination. But desired DNA fragment (about 1600 bp) bond also appeared in the result of migration. We picked up the fragment and extracted from gel.<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying in room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120914 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (50~60 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 13 hours.<br />
<br />
We succeeded to transform the cells but noticed that we have used wrong DNA solution. We'll try this DNA synthesis again tomorrow.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 15th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.<br />
<br />
Insert (RBS-phaB-RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|21 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(RBS-phaC-RBS-phaA-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120915 Digestion Insert-Vector.jpg|thumb|digestion result]]<br />
<br />
</p><br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying in room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120915 Ethapre Insert-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (50~60 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 17 hours.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 16th==<br />
<div><br />
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pT7-RBS-pSB1C3==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI, and pT7-RBS-pSB1C3 with EcoRI and SpeI. As a control, we digested pT7-RBS-pSB1C3 with only EcoRI to confirm the digestibility of This DNA (The digestibility with SpeI has confirmed in August 26th).<br />
<br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 35ng/ul)<br />
|41 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
Vector(pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg|thumb|digestion result]]<br />
<br />
We confirmed 4 bonds in the result of Inset DNA digestion. A Bond which shows the highest concentration and places about 8k ~ 10k bp area would be pBAD-RBS-eCFP-RBS-Ag43-pSTV28 construct. We considered about it bond whether remained from digestion, or at first formed dimer and separated by digestion. In either case, our target product has about 5200 bp and there is appropriate bond. We extracted the target bond from TBE gel and went next step.<br />
</p><br />
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying in room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA <br />
|4 ul<br />
|-<br />
|Vector DNA<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
Transformation ligation product into DH5&alpha;.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 13 hours.<br />
</p><br />
<br />
==Colony PCR of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(phaA-1083bp-F primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R down primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaC-RBS-phaA-RBS-phaB-dT-pSB1A2)as controls.<br />
Desired product is about 1800~2000bp.<br />
<br />
[[image:HokkaidoU2012 120916 coloP RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.<br />
</p><br />
<br />
==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 1 and No. 2 respectively). <br />
#Incubated at 37C for 20 hrs.<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_9
Team:HokkaidoU Japan/Notebook/aggregation Week 9
2012-09-26T20:08:23Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 27th==<br />
<div><br />
<br />
==Aggregation check==<br />
<p><br />
We found a lot of colonies on LBA plate, spreaded at August 26th.<br />
We incubated 3 colonies in LBAK solution including 1% L-arabinose for 18 hrs.<br />
However, we could not find expression of Ag43 protein.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
</p><br />
[[image:HokkaidoU2012_120827_take-iroiro.jpg|thumb|Plasmid extraction resulsts]]<br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120827 colop pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
The result did not show the band clearly. We selected No.1 and 2 colony for incubation.<br />
<br />
<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium.<br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 2 colonies (No.1 and No.2 respectively). <br />
#Incubated at 37C for 15 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2==<br />
<p><br />
#Mixed 1 ul DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours. <br />
</p><br />
<br />
==PCR of RBS-YFP-dT==<br />
<p><br />
Amplified the construct with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.2<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 28th==<br />
<div><br />
<br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by EcoRI and SpeI.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120828_pbad-rbs-ag43-dt-dig.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
<br />
==Plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3. We re-suspended No.1,2 colonies and incubated.<br />
<br />
[[image:HokkaidoU2012 120828 mini-prep pT7-RBS-Ag43-dT No.jpg|thumb|Plasmid extraction result]]<br />
<br />
</p><br />
<br />
==Estimation of Concentration of RBS-eYFP-dT and ptetR-pSB1A2==<br />
<p><br />
{|<br />
|Solution<br />
|Value (ul)<br />
|-<br />
|1kb ladder<br />
|1<br />
|-<br />
|Insert<br />
|1<br />
|-<br />
|Vector<br />
|1<br />
|-<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120828 Vector-ptetonpSB1C3-Concentrationcheck Insert-rbs-yfp-dt-PCR.jpg|thumb|Electrophoresis result]]<br />
<br />
<br />
From this result, We estimated that the concentration of Insert DNA solution is about 40 ng/ul, and Vector DNA is also about 40 ng/ul.<br />
</p><br />
<br />
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==<br />
<p><br />
ptet-pSB1A2(Vector) was digested with SpeI and PstI, and RBS-Ag43-dT(Insert) was cut with XbaI and PstI.<br />
<br />
<br />
<br />
<br />
Vector<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|5 ul<br />
|-<br />
|SpeI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Inset<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|14 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120828 Digestion Insert-rbs-yfp-dt-PCR(XP) Vector-ptetonpSB1C3(SP).jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that Insert and Vector DNA were digested.<br />
</p><br />
<br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for ptet-pSB1A2 and RBS-eYFP-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW. <br />
</p><br />
<br />
<br />
==Ligation==<br />
<p><br />
Ligation for ptet-pSB1A2 and RBS-eYFP-dT.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|3 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product (ptet-RBs-eYFP-dT on pSB1A2) into DH5&alpha;.<br />
#Mixed 1 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LBA.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hrs.<br />
</p><br />
<br />
<br />
==Gel extraction product check of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
We couldn't get desired plasmid DNA by transformation. We doubted contamination of non-digested vector DNA and decided to test the gel extraction product of pT7-RBS on pSB1C3 was successfully separated from non-digested product or not by transformation.<br />
#Mixed 1 ul of each DNA of ligation product and digestion product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 16 hours. <br />
<br />
<br />
There were no colony on the LBC plate that was spread solution mixed digestion product.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 29th==<br />
<div><br />
==Aggregation check==<br />
<p><br />
We incubated 9 colonies transformed at August 25th in LBAK solution including 1% L-arabinose for 16 hrs.<br />
Then we find that 1 culture expressing Ag43.<br />
Observed the E. coli cluster by a microscope of 100 magnifications.<br />
<br />
<br />
Then we checked whether pBAD promoter is behaving exactly or not.<br />
#1 colony ( could expressing Ag43 ) incubated in 2 ml LB at 37C for 2 hrs.<br />
#Prepared 5 kinds of LB medium differing the concentration of L-arabinose.<br />
<br />No. 1 :0.001%<br />
<br />No. 2 :0.1% <br />
<br />No. 3 :0.4%<br />
<br />No. 4 :1.0%<br />
<br />No. 5 :0%<br /><br />
#Added 400 ul of culture in several LB medium.<br />
#Incubated for 17 hrs and 30 min at 37C.<br />
<br />
</p><br />
<br />
==PCR of eCFP(E0020)==<br />
<p><br />
Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Desired product is about 800~900bp.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.2<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR of pT7-RBS-Ag43-dT on pSB1C3 transformed at August 28th. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp. This length is almost same as N2.<br />
<br />
[[image:HokkaidoU2012 120829 colop pT7-RBs-Ag43-dTonpSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We decided to incubate the No.7 and 8 colony.<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of pT7-RBS-Ag43-dTonpSB1C3 and Ag43-dTonpSB1AK3==<br />
<p><br />
Incubation of pT7-RBS-Ag43-dTonpSB1C3 (and Ag43-dT on pSB1AK3) in LBC (LBA) liquid medium.<br />
#Prepared 2 ml LBC (LBA) into culture tubes.<br />
#Re-suspended 2 colonies No.7 and No.8 respectively. Ag43-dT on pSB1AK3 was re-suspended the N2 colony. <br />
#Incubated at 37C for 17 hrs.<br />
</p><br />
<br />
<br />
==Estimation of Concentration of RBS-eYFP-dT (PCR product) and ptetR-pSB1A2==<br />
<p><br />
{|<br />
|Solution<br />
|Value (ul)<br />
|-<br />
|1kb ladder<br />
|1<br />
|-<br />
|Insert<br />
|1<br />
|-<br />
|Vector<br />
|1<br />
|-<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120829 PCR 100bpup-eCFP-200bpdown mini-prep B0034.jpg|thumb|Electrophoresis result]]<br />
<br />
<br />
From this result, We estimated that the concentration of Insert DNA solution is about 36 ng/ul, and Vector DNA is also about 29 ng/ul.<br />
</p><br />
<br />
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==<br />
<p><br />
ptet-pSB1A2(Vector) was digested with EcoRI and SpeI, and RBS-Ag43-dT(Insert) was cut with EcoRI and XbaI.<br />
And we used construct of pT7-RBS on pSB1C3 as control for the function of XbaI.<br />
<br />
<br />
<br />
Insert (eCFP)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (36 ng/ul)<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS on pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (29 ng/ul)<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBs on pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|100x BSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120829 Digestion eCFP RBSonpSB1A2 XbaIcontrol.jpg|thumb|digestion result]]<br />
<br />
From this result, Vector DNA were digested by XbaI, but remains some amount of non-digested DNA.<br />
And insert DNA solution were not existed in both d- and d+ lines.It means we failed to extract from gel after electrophoresis. We decided to retry PCR.<br />
</p><br />
<br />
==PCR of eCFP(E0020)==<br />
<p><br />
Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Desired product is about 800~900bp.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.0<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 30th==<br />
<div><br />
<br />
==Estimation of concentration of eCFP, pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT-pSB1AK3==<br />
<p><br />
No. 7,8 means the colony number of colony PCR of pT7-RBS-Ag43-dT-pSB1C3.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP mini-prep pT7-RBS-Ag43-dTonpSB1C3 No.jpg|thumb|electrophoresis result]]<br />
<br />
<br />
We failed the PCR for E0020.<br />
We estimated the concentration of pT7-RBS-Ag43-dT-pSB1C3 is 31 ng/ul and Ag43-dT-pSB1AK3 is 35 ng/ul.<br />
</p><br />
<br />
==Digestion of pT7-RBS-pSB1C3 and Ag43-dT-pSB1AK3==<br />
<p><br />
Digestion of pT7-RBS-pSB1C3 (as Vector) and Ag43-dT-pSB1AK3 (as Insert) to make pT7-RBs-Ag43-dT-pSB1C3 construct.<br />
<br />
Vector<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (31 ng/ul)<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xm buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Inset<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|14 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
As a background, we did PCR for eCFP again.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP digestion-Ag43-dTonpSB1AK3.jpg|thumb|digestion and PCR result]]<br />
<br />
<br />
We did not do digestion of vector DNA sokution, result, because it has only too low concentration to be digested product solution.<br />
<br />
<br />
We failed PCR again.<br />
The digestion result was confirmed that the Insert DNA was successfully digested by XbaI and SpeI.<br />
And we extracted the insert DNA digestion product from agarose gel by gel-extraction kit and got 20 ul of Insert DNA solution. <br />
</p><br />
<br />
==Digestion of pSB1AK3 by hindIII==<br />
<p><br />
The gel-extracted DNA solution contains Ag43-dT DNA and pSB1AK3 DNA because these DNA have same size of base pair and exist as same band in agarose gel. Thus we digest pSB1AK3 with HindIII to separate it from Ag43-dT.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|20 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|10<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
As a background, we did PCR for eCFP again.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP digestion(HindIII)-Ag43-dTonpSB1AK3.jpg|thumb|PCR and digestion result]]<br />
<br />
<br />
We failed the PCR for E0020.<br />
The digestion result was confirmed that the Insert DNA was successfully digested with HindIII.<br />
</p><br />
<br />
==Ethanol precipitation of pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT==<br />
<p><br />
We did ethanol precipitation to get more high concentration DNA solution of each part. And we got 5ul of each DNA solution.<br />
<br />
[[image:HokkaidoU2012 120831 Insert-d--d+ Vector-d+.jpg|thumb|ethanol precipitation result]]<br />
<br />
The electrophoresis result showed that the pT7-RBS-pSB1C3 construct was successfully digested by SpeI and get enough concentration of DNA solution to ligate. <br />
</p><br />
<br />
==Ligation of pT7-RBS-pSB1C3 and Ag43-dT==<br />
<p><br />
Ligation and pT7-RBS-pSB1C3 (as Vector) and Ag43-dT (as Insert).<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|2.5 ul<br />
|-<br />
|Insert DNA<br />
|2.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
[[image:HokkaidoU2012 120831 Ligation Vector Insert Ligationproduct(4ul).jpg|thumb|ligation result]]<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of Ag43-dT-pSB1AK3 and ptet-RBS-YFP-dT-pSB1A2==<br />
<p><br />
Incubation of Ag43-dT-pSB1AK3 and ptet-RBS-YFP-dT-pSB1A2 in LBA liquid medium.<br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 1 colony from each plate that each construct plated. <br />
#Incubated at 37C for 22 hrs (Ag43-dT-pSB1AK3 was Incubated for 41 hrs)<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 31st==<br />
<div><br />
==Plasmid extraction for pBAD-RBS-Ag43-dT on pSB1AK3==<br />
<p><br />
We used promega SV miniprep kit.<br />
We got 100 ul DNA solution.<br />
</p><br />
[[image:HokkaidoU2012_120831_pbad-rbs-ag43-dt-on1ak3.jpg|thumb|plasmid extraction result]]<br />
<br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by EcoRI and SpeI.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120831_take-iroiro2.jpg|thumb|digestion result]]<br />
<br />
==Transformation of pT7-RBS-Ag43-dT-pSB1C3 and eCFP (E0020)==<br />
<p><br />
#Mixed 2 ul ligation product and 1ul E0020 DNA solution to each 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA and LBC).<br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol (E0020 mixture skipped this step).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours.<br />
<br />
We failed to transformation of pT7-RBS-Ag43-dT-pSB1C3. <br />
</p><br />
<br />
==Confirmation of amplification of ptet-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
After plasmid extraction, we confirmed whether the desired construct ptet-RBS-eYFP-dT was really amplified or not by PCR.<br />
We chose EX-F primer as forward primer and PS-R primer as reverse primer then amplified about 1000bp of insert DNA.<br />
As controls, we chose DW as N1 and ptet-RBS-eCFP-dT construct as N2.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-F primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
[[image:HokkaidoU2012 120831 colop N1(DW) N2(ptet-RBS-eCFP-dT) 1(ptet-RBS-eYFP-dT).jpg|thumb|PCR result]]<br />
<br />
<br />
This image showed that the desired construct (about 1000 bp) amplified by incubation.<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of eCFP-pSB1A2==<br />
<p><br />
Incubation of eCFP-pSB1A2 in LBA liquid medium.<br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 1 colony. <br />
#Incubated at 37C for 16 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS-eYFP-dT-pSB1A2 and pT7-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
#Mixed 2 ul pT7-RBS-Ag43-dT-pSB1C3 ligation product and 1ul ptet-RBS-eYFP-dT-pSB1A2 DNA solution to each 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA and LBC).<br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol (ptet-RBS-eYFP-dT-pSB1A2 mixture skipped this step).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 1st==<br />
<div><br />
==Digestion for pSB1C3==<br />
<p><br />
We have to change the plasmid backbone to pSB1C3.<br />
We got the insert DNA at August 31th.<br />
So we need to get the pSB1C3 solution digested by EcoRI and SpeI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|3 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120902_take-iroiro.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of pBAD-RBS-Ag43-dT solution and pSB1C3 solution digested by EcoRI & SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for insert DNA and pSB1C3.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|1 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 1 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Plated 300 ul of the culture onto first LBC dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBC dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
</p><br />
<br />
==Plasmid extraction of Ag43-dT-pSB1AK3 and RBS-pSB1A2==<br />
<p><br />
We used plasmid extraction kit of Nippon genetics and got 50 ul Ag43-dT-pSB1AK3 plasmid DNA solution.<br />
</p><br />
<br />
==Digestion of eCFP-pSB1A2 and RBS-pSB1A2==<br />
<p><br />
To make a construct of eCFP-RBS-pSB1A2, we digested eCFP-pSB1A2 by EcoRI and SpeI, and RBS-pSB1A2 with EcoRI and XbaI. And we digested pT7-RBS-pSB1C3 by XbaI as a control for confirmation of the ability to digest.<br />
<br />
Insert (eCFP-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|11 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (29 ng/ul)<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|100x BSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120902 Digestion I-+ V-+ C(XbaI)-+.jpg|thumb|digestion result]]<br />
<br />
<br />
From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 2nd==<br />
<div><br />
==Ethanol precipitation of digestion products (eCFP and RBS-pSB1A2) and estimation of concentration==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120902 Ethapre V I.jpg|thumb|ethanol precipitation result]]<br />
<br />
We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 37 ng/ul.<br />
</p><br />
<br />
==Incubation of pT7-RBS-Ag43-dT-pSB1C3 transformation product==<br />
<p><br />
We failed to cultivation of above construct two times so we tried to incubation in LBC liquid medium.<br />
#Prepared 1.8 ml LBC into culture tubes.<br />
#Re-suspended 200 ul transformation product solution (which was mixed with LB and incubated for 3 hrs). <br />
#Incubated at 37C. <br />
</p><br />
<br />
==Ligation of eCFP and RBS-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (37 ng/ul)<br />
|1 ul<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|11 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of eCFP-RBS-pSB1A2==<br />
<p><br />
#Mixed 2 ul eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 19 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<br />
<div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_8
Team:HokkaidoU Japan/Notebook/aggregation Week 8
2012-09-26T20:07:39Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 20th==<br />
<div><br />
==Single colony isolation==<br />
<p><br />
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.<br />
#Picked up one colony.<br />
#Incubated on LBK (dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp up primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls.<br />
Desired product is about 300~400bp.<br />
<br />
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.<br />
</p><br />
<br />
<br />
==PCR==<br />
<p><br />
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Liquid culture==<br />
<p><br />
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBK (LBC) into culture tubes.<br />
#Resuspended 1 colonies. <br />
#Incubated the tubes at 37C for 16 hours (19 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 21st==<br />
<div><br />
==PCR==<br />
<p><br />
PCR of pBAD(containing araC)-RBS.<br />
And, we checked the plasmid which we refined by plasmid extraction at August 18th is pBAD-RBS on pSB1A3 or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-rbs_pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Aggregation check==<br />
<p><br />
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK.<br />
We checked the construct by induction of L-arabinose after 16 hours incubate.<br />
<br />
#2 ml of liquid culture divided two culture. (made two 1 ml culture)<br />
#Added 1 ml LBK in one culture as a negative control.<br />
#Added 900 ul LBK and 100 ul 20% L-arabinose.<br />
#Incubated at 37C 130 rpm for 2 hrs and 30 min.<br />
#Placed tubes on the table at 30 min.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used Plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
<br />
[[image:HokkaidoU2012 120821 pT7-RBS on pSB1C3 ColonyNo. 4,5 mini-prep.jpg|thumb|Plasmid extraction result]]<br />
<br />
<br />
The concentration of 20 ul of plasmid extraction products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2. <br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBA (LBC) into culture tubes.<br />
#Resuspended 2 colonies. <br />
#Incubated the tubes at 37C for 18 hours (16 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 22nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|plasmid extraction result]]<br />
<br />
<br />
<br />
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.<br />
We tried plasmid extraction to the latter, we got the 20 ul of DNA solution.<br />
And then, we did electrophoresis the plasmid extraction products and (pBAD-RBS and pBAD) gel extract products.<br />
<br />
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]<br />
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR of pT7-RBS on pSB1C3.<br /><br />
<br />
We used 4 kinds of primer set.<br />
<br /><br />
1 : EX-F , PS-R primer<br /><br />
2 : EX-F , 200b down primer<br /><br />
3 : 100b up , PS-R primer<br /><br />
4 : 100b up , 200b down primer<br /><br />
The density of primer solutions is 10 uM.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer<br />
|1 ul<br />
|-<br />
|Reverse Primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 23rd==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3.<br />
<br />
Digestion of pBAD-RBS.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pBAD-RBS (100 ng/ul)<br />
|12 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Digestion of Ag43-dT on pSB1AK3.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT (120 ng/ul)<br />
|7 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120824_ag43-dt-dig2.jpg|thumb|Ag43-dT digestion result]]<br />
[[image:HokkaidoU2012_120824_pbad-rbs-dig.jpg|thumb|pBAD-RBS digestion result]]<br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
</p><br />
==Digestion==<br />
<p><br />
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 257ng/ul)<br />
|9 ul<br />
|-<br />
|HindIII(15 U/ul)<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]<br />
<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained as non-digested products.<br />
<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 24th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.<br />
<br /><br />
<br /><br />
Ag43-dT on pSB1AK3<br />
<br /><br /><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pBAD-RBS<br /><br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 20ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]<br />
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.<br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|4 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 25th==<br />
<div><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and electrophoresis result image of ligation products, then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligated DNA solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. <br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|600 (10 hours)<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 26th==<br />
<div><br />
<br />
===Ligation===<br />
<p><br />
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS<br />
|3 ul<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Plated 300 ul of the culture onto first LBA dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.<br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|0.25 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|DW<br />
|1.75 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_7
Team:HokkaidoU Japan/Notebook/aggregation Week 7
2012-09-26T20:07:12Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 16th==<br />
<div><br />
==ligation==<br />
<p><br />
We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS (5 ng/ul)<br />
|1 ul<br />
|-<br />
|Ag43-dT (25 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Ag43-dT on pSB1AK3 digestion with SpeI and XbaI.<br />
<br />
Ag43-dT<br />
SpeI and XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
[[image:HokaidoU2012_120813_pbad-rbs-ori-x10.jpg|thumb|pBad-RBS on pSB1A3 electrophoresis result]]<br />
[[image:HokkaidoU2012_120813_ag43-dt-s-x.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 17th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1 colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls.<br />
Desired product is about 800~1000bp.<br />
<br />
[[image:HokkaidoU2012_120817_pbad-rbs-ag43-dt-coloP.jpg|thumb|Colony PCR result]]<br />
<br />
From this result, N2 has about 500bp band.<br />
We use to liquid culture, No. 7,10,12.<br /><br />
We cultured these DNA in E. coli solution, after added 200 ul LB, at 37C for 3 hrs.<br />
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for hours at 37C.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of BBa_I13453 (pBAD on pSB1A3) into DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 19 hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
From sequencing results, we found that we failed to make pT7-RBS on pSB1K3.<br /><br />
So, we tried to make it again. <br /><br />
When the restriction enzyme digest the DNA, it is important for 3A assembry to be digested the target plasmid completely. So, we activated the restriction enzymes for 10 hours.<br /><br />
<br /><br />
pT7 (BBa_I719005)<br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
RBS (BBa_B0034)<br />
XbaI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
pSB1C3<br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (25 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 18th==<br />
<div><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for three digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Before digestion of pBAD-RBS on pSB1A3 and Ag43-dT on pSB1AK3, it is necessary to refine these two DNA. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 20 ul of DNA solution from 10 ul of plasmid extraction products.<br />
</p><br />
==Digestion==<br />
<p><br />
Digested pBAD-RBS on pSB1A3 as insert and Ag43-dT on pSB1AK3 as vector with EcoRI and SpeI to make a constract, pBAD-RBS-Ag43-dT on pSB1AK3.<br /><br />
<br /><br />
<br />
pBAD-RBS<br /><br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|plasmid extraction product (20 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT on pSB1AK3<br /><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Gel extraction product (40 ng/ul)<br />
|8 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|17 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012_120818_pbad-rbs--ag43-dt-dig.jpg|thumb|Digestion result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for colony number of No. 10 and 12 of pBAD-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
[[image:HokkaidoU2012_120818_pbad-rbs-ag43-dt.jpg|thumb|plasmid extraction result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for pT7 and RBS as inserts and pSB1C3 as vector.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7 (100 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT (100 ng/ul)<br />
|2 ul<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS (80 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT on pSB1AK3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 19th==<br />
<div><br />
==Transformation==<br />
<p><br />
Transformation of ligation product (two construct ligated at August 18) into DH5&alpha;.<br /><br />
<br />
<br />
pT7-RBS on pSB1C3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBC.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 18 hours.<br />
<br />
pBAD-RBS-Ag43-dT on pSB1AK3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LBK.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 16 hours and 30 minutes.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR for pT7-RBS on pSB1K3.<br />
By sequencing reaction, we found the No. 10 plasmid is not pT7-RBS on pSB1K3.<br />
We checked the other plasmid is the same or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-Forward primer : 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-Reverse primer : 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|30<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 45<br />
<br />
[[image:HokkaidoU2012_120819_pcr.jpg|thumb|PCR result]]<br />
<br />
No. 4 plasmid has independent bands.<br />
However, if it is pT7-RBS on pSB1K3, it has about 60~70bp band.<br />
<br />
</p><br />
<br />
</div><div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_6
Team:HokkaidoU Japan/Notebook/aggregation Week 6
2012-09-26T20:06:34Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 6th==<br />
<div><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|1 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012_120806_pBad-rbs.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB then incubated the cells for 2 hours at 37C.<br />
#Prepared and Labeled two LBK plates.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 900 ul of LB to 100 ul of the culture and plated 300 ul of it onto second dish and spread. <br />
#Incubated them at 37C for 16 hours.<br />
<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for plasmid extraction product (pBad-RBS). Because the refine of plasmid extraction product is not enough. <br /><br />
We used 15 ul of all solution.<br />
#Added 1.5 ul of NaoAc(3 M), 1.5 ul of glycogen and 38 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of ethanol precipitation product(pBad-RBS).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
</p><br />
[[image:HokkaidoU2012_120807_pbad-rbs-digested.jpg|thumb|digestion result]]<br />
<br />
From this result, speI digested DNA completely.<br /><br />
So I think that the pT7-RBS on pSB1K3 DNA solution is not pure.<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 7th==<br />
<div><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43 on pSB1C3 (K346007)) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012_120807_colonyPCR.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies of No. 1 and 2 and 5 and 11 and 14 have deep band.<br />
Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours at 37C.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 8th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of colony No. 1 and 14. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012_120808_pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
<br />
And then, we did plasmid extraction for colony No. 2,5,11. The results of them are the same.<br />
</p><br />
<br />
==Sequencing==<br />
<p><br />
PCR for sequencing.<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To purify the PCR product, we did ethanol precipitation.<br />
<br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
Then ran a sequencing machine (ByoDye Terminator v3.1 Cycle Sequencing Kit)<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 9th==<br />
<div><br />
==Ligation==<br />
<p><br />
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br /><br />
We did ligation (pT7-RBS on pSB1K3 and Ag43-dT) and transformation it, at August 6th.<br />
However, we could not get the target plasmid. (pT7-RBS-Ag43-dT on pSB1K3)<br />
So, we did ligation by using more Insert DNA part.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|1 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br /><br />
Transformation of August 6th, we found only 14 colonies. So this time, we use 3 ul of DNA solution for transformation.<br />
<br />
#Added 3 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 400 ul of LB then incubated the cells for 2 hours at 37C.<br />
#Prepared and Labeled two LBK plates.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread. <br />
#Incubated them at 37C for 16 hours.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 11st==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU1120811 coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies of No. 6 and 8 is like N2 band.<br />
Next step, we resuspended these 2 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 16 hours at 37C.<br />
<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 12nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of No. 6 and 8. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012_120812_pt7-dt.jpg|thumb|plasmid extraction result]]<br />
</p><br />
<br />
</div><div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_5
Team:HokkaidoU Japan/Notebook/aggregation Week 5
2012-09-26T20:06:02Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 30th==<br />
<div><br />
==Digestion==<br />
<p><br />
I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.<br />
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
I think that the E. coli which we used transformation is BL21(DE3)pLysS.<br />
So, the E. coli could grow on LBC plate.<br /><br />
I'm going to do transformation into DH5&alpha;.<br />
<br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 200 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish. <br />
#Incubated the plates at 37C for 14 hrs.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 31st==<br />
<div><br />
==Liquid culture==<br />
<p><br />
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 1st==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT which had been incubated from 31st July. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012 120801 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br />
<br /><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''pT7-RBS(30 ng/ul) '''=No. 10<br />
<br /><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012 120801 pt7-rbs-digS.jpg|thumb|digestion result]]<br />
<br />
We couldn't digested them exactly, so we tried to digest once more time.<br /><br />
<br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br /><br />
SpeI 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for pBAD-RBS on pSB1K3.<br />
#Added 2 ml of LBK into culture tube.<br />
#Scraped the surface of glycerol stock of construct.<br />
#Incubated the tube at 33C.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 2nd==<br />
<div><br />
<p><br />
==Preparing chemical competent cell==<br />
Preparing chemical competent cell of BL21, JM109 and DH5&alpha;.<br />
Chemical competent cell made in each E. coli strains.<br />
<br /><br /><br />
Our competent cell Protocol<br />
#Did single colony isolation on LB plate.<br />
#Incubated the plate for 15-19 hours at 37C.<br />
#Lifted colony of E. coli into 2 ml LB.<br />
#Incubated at 37C for 12-16 hrs at 180-200 rpm.<br />
#Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.<br />
#Incubated cells at 20C (over 24 hrs) at 140 rpm.<br />
#Selected culture by measuring OD 600.<br />
#Incubated the 300 ml flask for 10 min on ice.<br />
#Transfered the culture into two 50 ml Falcon tubes.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)<br />
#Collected them to one tube.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 3.2 ml of TB.<br />
#Instilled 0.24 ml of DMSO in precipitant.<br />
#Incubated the 50 ml Falcon tube for 10 min on ice.<br />
#Divided 50 ul of solutions in each 0.5 ml tubes.<br />
#Freezed the suspension by liquid nitrogen.<br />
#Stored at –80C.<br />
<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)<br />
<br />
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|Electrophoresis result]]<br />
There were low concentration band above thick band. This thick band was same as digestion minus band.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 3rd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for the result of digestion.<br />
[[image:HokkaidoU2012 120803 pt7-rbs-dspe1.jpg|thumb|Electrophoresis result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Spread 300 ul of the transformation onto first dish.<br />
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish. <br />
#Incubated the plates at 37C for 15 hrs 45 min.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm Ag43-f4 primer.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-f4 primer<br />
|1 ul<br />
|-<br />
|PS-R primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120803 Ag43 PCR(Forward=ag43-f4 Reverse=PS-R).jpg|thumb|PCR result]]<br />
<br />
From this result, we could see that the ag43-f4 primer had worked and DNA of last 500~600 bp of ag43 to BioBrick suffix were increased.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 4th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012 120804 pt7-rbs-ag43-dt coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band.<br />
Next step, we resuspended these three colonies and incubated. <br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture of colonies passed the colony PCR test.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended 3 colonies.<br />
#Incubated the tubes at 37C for 13 hrs. <br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 5th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
[[image:HokkaidoU2012 120805 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.<br />
<br />
<br />
pT7-RBS on pSB1K3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS (once digestioned with SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 pT7-RBS digestion SpeI (no.jpg|thumb|digestion result]]<br />
From this result, the bottom band was digested incorrectly and middle band was digested correctly, we thought. Was the above band something contaminated in the DNA solution?<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction of middle band. We thought this band would be the exactly digested DNA fragment. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
==Sequencing==<br />
<p><br />
Sequencing to confirm what kind of DNA we made.<br />
<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To extract the PCR product, we did ethanol precipitation.<br />
<br /><br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di. <br />
<br />
<br />
Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)<br /><br />
We could not get the sequencing data.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 Ag43-dT on pSB1AK3(d+ with XbaI &amp; SpeI) digestion with HindIII.jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_7
Team:HokkaidoU Japan/Notebook/aggregation Week 7
2012-09-26T20:02:28Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 16th==<br />
<div><br />
==ligation==<br />
<p><br />
We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS (5 ng/ul)<br />
|1 ul<br />
|-<br />
|Ag43-dT (25 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Ag43-dT on pSB1AK3 digestion with SpeI and XbaI.<br />
<br />
Ag43-dT<br />
SpeI and XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
[[image:HokaidoU2012_120813_pbad-rbs-ori-x10.jpg|thumb|pBad-RBS on pSB1A3 electrophoresis result]]<br />
[[image:HokkaidoU2012_120813_ag43-dt-s-x.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 17th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1 colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls.<br />
Desired product is about 800~1000bp.<br />
<br />
[[image:HokkaidoU2012_120817_pbad-rbs-ag43-dt-coloP.jpg|thumb|Colony PCR result]]<br />
<br />
From this result, N2 has about 500bp band.<br />
We use to liquid culture, No. 7,10,12.<br /><br />
We cultured these DNA in E. coli solution, after added 200 ul LB, at 37C for 3 hrs.<br />
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for hours at 37C.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of BBa_I13453 (pBAD on pSB1A3) in DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 19 hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
From sequencing results, we found that we failed to make pT7-RBS on pSB1K3.<br /><br />
So, we tried to make it again. <br /><br />
When the restriction enzyme digest the DNA, it is important for 3A assembry to be digested the target plasmid completely. So, we activated the restriction enzymes for 10 hours.<br /><br />
<br /><br />
pT7 (BBa_I719005)<br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
RBS (BBa_B0034)<br />
XbaI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
pSB1C3<br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (25 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 18th==<br />
<div><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for three digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Before digestion of pBAD-RBS on pSB1A3 and Ag43-dT on pSB1AK3, it is necessary to refine these two DNA. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 20 ul of DNA solution from 10 ul of plasmid extraction products.<br />
</p><br />
==Digestion==<br />
<p><br />
Digested pBAD-RBS on pSB1A3 as insert and Ag43-dT on pSB1AK3 as vector with EcoRI and SpeI to make a constract, pBAD-RBS-Ag43-dT on pSB1AK3.<br /><br />
<br /><br />
<br />
pBAD-RBS<br /><br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|plasmid extraction product (20 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT on pSB1AK3<br /><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Gel extraction product (40 ng/ul)<br />
|8 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|17 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012_120818_pbad-rbs--ag43-dt-dig.jpg|thumb|Digestion result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for colony number of No. 10 and 12 of pBAD-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
[[image:HokkaidoU2012_120818_pbad-rbs-ag43-dt.jpg|thumb|plasmid extraction result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for pT7 and RBS as inserts and pSB1C3 as vector.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7 (100 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT (100 ng/ul)<br />
|2 ul<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS (80 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT on pSB1AK3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 19th==<br />
<div><br />
==Transformation==<br />
<p><br />
Transformation of ligation product (two construct ligated at August 18) in DH5&alpha;.<br /><br />
<br />
<br />
pT7-RBS on pSB1C3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBC.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 18 hours.<br />
<br />
pBAD-RBS-Ag43-dT on pSB1AK3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LBK.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 16 hours and 30 minutes.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR for pT7-RBS on pSB1K3.<br />
By sequencing reaction, we found the No. 10 plasmid is not pT7-RBS on pSB1K3.<br />
We checked the other plasmid is the same or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-Forward primer : 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-Reverse primer : 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|30<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 45<br />
<br />
[[image:HokkaidoU2012_120819_pcr.jpg|thumb|PCR result]]<br />
<br />
No. 4 plasmid has independent bands.<br />
However, if it is pT7-RBS on pSB1K3, it has about 60~70bp band.<br />
<br />
</p><br />
<br />
</div><div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_5
Team:HokkaidoU Japan/Notebook/aggregation Week 5
2012-09-26T20:01:38Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 30th==<br />
<div><br />
==Digestion==<br />
<p><br />
I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.<br />
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
I think that the E. coli which we used transformation is BL21(DE3)pLysS.<br />
So, the E. coli could grow on LBC plate.<br /><br />
I'm going to do transformation , use DH5&alpha;.<br />
<br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 200 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish. <br />
#Incubated the plates at 37C for 14 hrs.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 31st==<br />
<div><br />
==Liquid culture==<br />
<p><br />
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 1st==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT which had been incubated from 31st July. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012 120801 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br />
<br /><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''pT7-RBS(30 ng/ul) '''=No. 10<br />
<br /><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012 120801 pt7-rbs-digS.jpg|thumb|digestion result]]<br />
<br />
We couldn't digested them exactly, so we tried to digest once more time.<br /><br />
<br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br /><br />
SpeI 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for pBAD-RBS on pSB1K3.<br />
#Added 2 ml of LBK into culture tube.<br />
#Scraped the surface of glycerol stock of construct.<br />
#Incubated the tube at 33C.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 2nd==<br />
<div><br />
<p><br />
==Preparing chemical competent cell==<br />
Preparing chemical competent cell of BL21, JM109 and DH5&alpha;.<br />
Chemical competent cell made in each E. coli strains.<br />
<br /><br /><br />
Our competent cell Protocol<br />
#Did single colony isolation on LB plate.<br />
#Incubated the plate for 15-19 hours at 37C.<br />
#Lifted colony of E. coli into 2 ml LB.<br />
#Incubated at 37C for 12-16 hrs at 180-200 rpm.<br />
#Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.<br />
#Incubated cells at 20C (over 24 hrs) at 140 rpm.<br />
#Selected culture by measuring OD 600.<br />
#Incubated the 300 ml flask for 10 min on ice.<br />
#Transfered the culture into two 50 ml Falcon tubes.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)<br />
#Collected them to one tube.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 3.2 ml of TB.<br />
#Instilled 0.24 ml of DMSO in precipitant.<br />
#Incubated the 50 ml Falcon tube for 10 min on ice.<br />
#Divided 50 ul of solutions in each 0.5 ml tubes.<br />
#Freezed the suspension by liquid nitrogen.<br />
#Stored at –80C.<br />
<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)<br />
<br />
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|Electrophoresis result]]<br />
There were low concentration band above thick band. This thick band was same as digestion minus band.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 3rd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for the result of digestion.<br />
[[image:HokkaidoU2012 120803 pt7-rbs-dspe1.jpg|thumb|Electrophoresis result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Spread 300 ul of the transformation onto first dish.<br />
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish. <br />
#Incubated the plates at 37C for 15 hrs 45 min.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm Ag43-f4 primer.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-f4 primer<br />
|1 ul<br />
|-<br />
|PS-R primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120803 Ag43 PCR(Forward=ag43-f4 Reverse=PS-R).jpg|thumb|PCR result]]<br />
<br />
From this result, we could see that the ag43-f4 primer had worked and DNA of last 500~600 bp of ag43 to BioBrick suffix were increased.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 4th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012 120804 pt7-rbs-ag43-dt coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band.<br />
Next step, we resuspended these three colonies and incubated. <br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture of colonies passed the colony PCR test.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended 3 colonies.<br />
#Incubated the tubes at 37C for 13 hrs. <br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 5th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
[[image:HokkaidoU2012 120805 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.<br />
<br />
<br />
pT7-RBS on pSB1K3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS (once digestioned with SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 pT7-RBS digestion SpeI (no.jpg|thumb|digestion result]]<br />
From this result, the bottom band was digested incorrectly and middle band was digested correctly, we thought. Was the above band something contaminated in the DNA solution?<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction of middle band. We thought this band would be the exactly digested DNA fragment. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
==Sequencing==<br />
<p><br />
Sequencing to confirm what kind of DNA we made.<br />
<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To extract the PCR product, we did ethanol precipitation.<br />
<br /><br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di. <br />
<br />
<br />
Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)<br /><br />
We could not get the sequencing data.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 Ag43-dT on pSB1AK3(d+ with XbaI &amp; SpeI) digestion with HindIII.jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_4
Team:HokkaidoU Japan/Notebook/aggregation Week 4
2012-09-26T20:00:52Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 23rd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for Ag43(resuspended colony incubated at July 17th and resuspended colony incubated at July 20th). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 24th==<br />
<div><br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for Ag43(plasmid extraction at July 23rd) and Ag43 digestion results(digested with EcoRI and SpeI)<br />
<br />
[[image:HokkaidoU2012 120724 ag43-dt pcr.jpg|thumb|Plasmid extraction result]]<br />
<br />
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product.jpg|thumb|Digestion result]]<br />
<br />
From this digestion result, we found out that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use for digestion.<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW. <br />
<br />
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]]<br />
<br />
From this result, we estimated that the concentration of ethanol precipitation product is about 40 ng/ul.<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion to confirm how many PstI cutting sites are there in K346007 and for Ag43-dT complex with SpeI and XbaI.<br />
<br />
<br /><br />
Ag43<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br /><br />
SpeI and XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|12 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion results.<br />
<br />
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S then cut with P).jpg|thumb|Ag43 d+(P) Digestion result]]<br />
[[image:HokkaidoU2012 120724 ag43-dplus.jpg|thumb|Ag43-dT d+(X&S) Digestion result]]<br />
<br />
From this result, we found that '''there are 6 PstI cutting sites in K346007(Ag43)'''. <br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction of Ag43-dt digestion result. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Ag43-dT and pSB1AK3 mixture solution(each DNA fragment is about 3kbp) digested with HindIII to digest pSB1AK3.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 25th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 with SpeI.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012 120725 Digestion Ag43-dt on pSB1AK3(HindIII) &amp; pT7-RBS on pSB1K3(SpeI).jpg|thumb|Digestion result(Ag43-dT and pT7-RBS)]]<br />
<br />
We were confirmed that pSB1AK3 was digested and became 1.3k and 1.8k bp fragments by HindIII. <br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI).<br />
We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation of digestion and gel extraction products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 5 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert).<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 26th==<br />
<div><br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into BL21.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Spread 200 ul of the transformation onto first dish.<br />
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish. <br />
#Incubated the plates at 37C for over 30 hrs.<br />
<br />
There were no colony on the plates.<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis of digestion and ligation products.<br />
#Placed TBE agarose gel in Electrophoresis chamber.<br />
#Added 1/2X TBE buffer to Electrophoresis chamber.<br />
#Added 5 ul of EtBr and ran at 100 V for 30 min. <br />
#Apply 1kb DNA ladder and each samples.<br />
#Ran at 100 V for 30 min.<br />
<br />
[[image:HokkaidoU2012 120726 digestion ligation.jpg|thumb|Digestion and Ligation results]]<br />
There are no band in the lane of ligation products. But if digestion products were not ligated, two bands of digestion products would exist in the lane. <br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 27th==<br />
<div><br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into BL21.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled three petri dishes with LBK X2 and LBC.<br />
#Spread 300 ul of the transformation onto LBK dish.<br />
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto LBC dish and LBK dish. <br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 28th==<br />
<div><br />
<p><br />
[[image:HokkaidoU2012 120728 Transformation result.jpg|thumb|Transformation result left:LBC center:LBK right:LBK]]<br />
There were many colonies on LBC. We guessed we mistook pT7-RBS on pSB1C3 for pT7-RBS on pSB1K3.<br />
</p><br />
==Liquid culture==<br />
<p><br />
Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.<br />
#Added 2 ml of LBC into culture tubes.<br />
#Resuspended 5 colonies.<br />
#Incubated the tubes at 30C for 22 hrs.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 with SpeI and Ag43-dT on pSB1AK3(cut with SpeI & XbaI) with HindIII.<br />
<br /><br />
pT7-RBS on pSB1K3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|} <br />
<br />
Ag43-dT on pSB1AK3(cut with SpeI & XbaI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
Digested at 37C for 2 hrs.<br />
<br />
[[image:HokkaidoU2012 120728 Digestion pT7-RBS on pSB1K3(SpeI) Ag43-dT on pSB1AK3(HindIII).jpg|thumb|digestion result]]<br />
<br />
This results showed that pSB1AK3 was successfully digested into fragments(1.3K and 1.8K bp), but we couldn't confirm whether pT7-RBS on pSB1K3 was digested or not.<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution. <br />
</p><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dryed out at room temperature after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 29th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for 5 cultures of pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
[[image:HokkaidoU2012_120729_pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction products]]<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
We need to confirm the DNA is pT7-RBS-Ag43-dT or not.<br />
<br />
<br />
Plasmid extraction products(pT7-RBS-Ag43-dT)<br /><br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Plasmid extraction products(pT7-RBS-Ag43-dT)<br /><br />
EcoR1 and Spe1<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoR1<br />
|1 ul<br />
|-<br />
|Spe1<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion results.<br />
<br />
[[image:HokkaidoU2012_120729_ag43-dig.jpg|thumb|Digestion result]]<br />
<br />
I could not understand what is happend.<br />
I tried it again, but the result was the same.<br />
<br />
</p><br />
<br />
</div><div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_3
Team:HokkaidoU Japan/Notebook/aggregation Week 3
2012-09-26T20:00:09Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 16th==<br />
<div><br />
Ag43, dT<br />
===Electrophoresis===<br />
<p><br />
Results of digestion at 15th.<br />
<br />
[[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&amp;P-1.jpg|thumb|Digestion result image]]<br />
Product:Ag43(K346007)=3120bp and 2070bp<br />pT7-RBS on pSB1K3=2247bp<br />
<br /><br />
We confirmed there are some kinds of restriction enzyme site in K346007 (digested with SpeI, PstI), and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.<br />
</P><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. Used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution.<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digested and extracted products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW.<br />
</p><br />
<br />
<br /><br />
--------<br />
<br /><br />
<br />
dT(B0015) would be amplified incorrectly and we couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.<br />
<br />
===PCR===<br />
<p><br />
PCR for dT(B0015)<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp_up forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(200bp_down Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]<br />
<br />
We migrated B0015 of plasmid extraction product, digestion product, and PCR product.<br />
dT done PCR would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
'''Digestion for dT which amplified with PCR.'''<br />
Digested with XbaI and PstI.<br />
<br /><br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
----<br />
<br />
Ag43<br />
<br />
'''Digestion result of Ag43 was incorrect'''. We digested Ag43 once more time.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for Ag43 with SpeI and PstI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43 DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120716 Ag43d- Ag43d+.jpg|thumb|Digestion result image]]<br />
<br />
There are same results with digestion result of recent. '''We thought PstI would cut different site'''.<br />
What is this 500bp fragment????<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50ul of DNA solution.<br />
</p><br />
<br />
----<br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.<br />
<br />
'''Colonies which have Ag43-dT on pSB1T3 were closely existed so we would picked up two or more colonies.''' <br />
#Picked up one (or tow?) colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBC and LBT.<br />
#Incuvated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 17th==<br />
<div><br />
<p><br />
Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3<br />
</p><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products incuvated from yesterday(16th).<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And plasmid extraction products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated.<br />
<br />
<br />
If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp.<br />
And if ligation and transformation, plasmid extraction were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp. <br />
<br />
[[image:HokkaidoU2012 120717 Ag43-Ag43d+(E&amp;S)-(Ag43-dT on pSB1T3)-(pT7-RBS on pSB1C3).jpg|thumb|Digestion and plasmid extraction result image]]<br />
<br />
From this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. '''would PstI not work correctly?''' <br />
<br />
<br />
'''We planed to use dT as vector and Ag43 as Insert.''' A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.<br />
<br />
<br />
About plasmid extraction products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought '''which means we failed single colony isolation then resuspended two another E.coli colonies''' another ligated DNA were transformed. <br />
In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.<br />
<br />
<br />
To confirm plasmid extraction products were really ligated correct DNA fragments, first we extracted Ag43-dT on pSB1T3(low bp band) from TBE gel and digested with EcoRI and PstI.<br />
</p><br />
<br />
----<br />
'''dT Vector plasmid change'''<br />
To use dT as a vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.<br />
<br />
==Digestion==<br />
<p><br />
Digestion to change the plasmid backbone.<br />
Used DNA solution as PCR product(did at 16th) and digestioned pSB1T3 were already exist.<br /><br />
<br /><br />
<br />
Digestion mix<br /><br /><br />
<br />
<br />
double digestion(EcoRI and PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717-dt-digestion-print.jpg|thumb|Digestion results]]<br />
<br />
We couldn't confirm digestion results.<br />
<br />
<br />
</p><br />
<br />
----<br />
pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3<br />
<br />
==Electrophoresis and gel extraction==<br />
<p><br />
[[image:HokkaidoU2012 120717-ag43-dt-psb1t3.jpg|thumb|plasmid extraction result]]<br />
'''Gel extraction for Ag43-dT on pSB1T3.'''<br /><br />
We cut low bp band (see image below).<br />
Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not.<br />
<br /><br /><br />
<br />
'''Ag43-dT on pSB1T3(30 ng/ul)'''<br />
<br /><br /><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br /><br />
'''pT7-RBS on pSB1C3(40 ng/ul)'''<br />
<br /><br /><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717 Ag43 dT(d-,d+E,d+P.jpg|thumb|Digestion result]]<br />
<br />
From this digestion results, <br />
*Cut with EcoRI,PstI and E&P(EcoRI and PstI) showed different base pair bands compared with d-(digestion minus).<br />
*Cut with E&P showed two DNA bands: about 2000bp and 500~1000 bp.<br />
*All of digestion products existed lower place than correct band.Correct DNA would show about 5000bp(EcoRI and PstI), 3000bp and 2000bp (E&P). <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 18th==<br />
<div><br />
<br />
<br />
'''We decided change plasmid backbone of dT pSB1AK3 to pSB1T3.'''<br /><br />
If we cut Ag43-dT on pSB1AK3 with EcoRI and PstI to confirm insert DNA, DNA fragment base pair resemble each other(about 3000bp), so then we can't identify them.<br />
<br />
===digestion===<br />
<p><br />
<br />
'''Digestion to confirm what kind of restriction enzyme sites Ag43(K346007) has.'''<br />
<br /><br /><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|- <br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120718-k346007-digestion print.jpg|thumb|Digestion result]]<br />
From this result, we doubted '''are there one or more another PstI resutriction enzyme site except BioBrick suffix'''?<br />
</p><br />
<br />
===PCR===<br />
<p><br />
PCR to confirm what DNA fragment is ligated to vector as insert.<br />
We used PCR for pT7-RBS on pSB1C3. So the result would be 80~90bp band.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Biobrick prefix forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(Biobrick suffix Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
[[image:HokkaidoU2012 120718 pT7-RBS on pSB1C3 EX-F PS-R PCR.jpg|thumb|PCR product]]<br />
</p><br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated(19hrs incubated) Ag43-dT on pSB1T3.<br />
<br />
#Prepared 2 ml LB.<br />
#Added Tet.<br />
#Resuspended one colony.<br />
#Incuvated 16 hrs.<br />
</p><br />
<br />
----<br />
We decided to start another ligation plan. First we digestion Ag43(K346007) and dT(B0015) with EcoRI&SpeI and EcoRI&XbaI then ligate. Ligation product cut with XbaI&SpeI and HindiIII. HindiIII can cut the site which is exist in pSB1AK3 then we can confirm Ag43-dT(about 3000bp) and pSB1K3(3000bp: cutting fragment will be about 1000bp). Next we digest pT7-RBS on pSB1C3 with SpeI only and ligate X-Ag43-dT-S and S-pSB1C3-pT7-RBS-S. If that go well, we can get pT7-RBS-Ag43-dT on pSB1C3 plasmid DNA which has never been digested with PstI. <br />
<br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(K346007)<br />
</p><br />
</div><br />
<br />
==July 19th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and Ag43(K346007) which is inserted in pSB1C3.<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</P><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for Ag43-dT on pSB1T3[about 5400bp] and Ag43(K346007)[about 5200bp] plasmid extraction products.<br />
<br />
[[image:HokkaidoU2012 120719 Ag43-dT on pSB1T3 and Ag43(K346007) mini-prep product.jpg|thumb|plasmid extraction result]]<br />
<br />
From this plasmid extraction result, Ag43-dT on pSB1T3 showed obviously higher DNA band than we estimated. It means '''Ag43-dT and pSB1T3 were misligated.''' And Ag43(K346007), we thought '''Ag43(K346007) DNA band existed in correct area''' because this DNA has about 5200bp and plasmid DNA migrates more far than linear DNA. Additionally, one weak 10kbp band existed in Ag43(K346007) lane.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
We conducted two digestion.<br />
*Digestion for Ag43(K346007) cut with EcoRI and SpeI. This DNA is for confirmation of PstI restriction enzyme cutting site.<br />
*Digestion for Ag43(K346007) and dT(B0015) cut with EcoRI & SpeI and EcoRI & XbaI.<br />
But we cut dT PCR product which didn't have plasmid vector so we retry digestion of dT after.<br />
<br />
<br /><br /><br />
Insert Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|16 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Vector dT(E&X)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT(EcoRI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
dT(XbaI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7.8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120719 Ag43 cut with E&amp;S(each enzyme 0.1ul) and Ag43 cut with E&amp;S(each enzyme 1ul).jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed Ag43(K346007) was successfully digested into two fragments and low concentration of restriction enzyme cause unperfectly cut of plasmid extraction product. But there are correct two bands in Ag43 cutting result so restriction enzyme worked. And about dT digestion, XbaI worked in halfway. We thought this is because we didn't added BSA buffer into dT digestion mix.<br />
<br />
<br />
</p><br />
<br />
And we retried digestion of dT which was plasmid extraction and gel extracted products. Recipe was same as above(E&X).<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 20th==<br />
<div><br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion result done yesterday(dT cut with EcoRI and XbaI).<br />
Added 5 ul of EtBr and Pre-migrated for 30 min.<br />
Additionaly, to confirm the concentration of Ag43(K346007) gel extract result.<br />
<br />
[[image:HokkaidoU2012 120720 dTd- dTd+ Ag43(cut with E&amp;S)gelextract.jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed dT was successfully cut with EcoRI & SpeI.<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extraction products above.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 5 ul of DW.<br />
</P><br />
<br />
==Ligation==<br />
<p><br />
We ligated Ag43(purified at 7/17 and 7/20) as an insert and dT as vector.<br />
<br />
<br /><br /><br />
Ag43(7/20) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|2 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(7/17) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|4 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 21st==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion and ligation products yesterday.<br />
<br />
<br />
[[image:HokkaidoU2012 120721 ag43-d+ dt-d+ ligation print.jpg|thumb|Digestion and Ligation results]]<br />
<br />
There are three bands in ligation products(Ag43(7/20) + dT(on pSB1AK3))). Lower band would be digestion result which couldn't be ligated with dT. Middle band would be successfully ligaed DNA which have about 6k bp(Ag43 has 3.1k bp and dT on pSB1AK3 has 3.2k bp). And higher band would make something dimer, we thought. <br />
<br />
</p><br />
<br />
==Single colony isolation==<br />
<p><br />
Single colony isolation for incubated colonies spread yesterday. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extracted Ag43(E&S) gel extraction product for digestion of PstI Star activity confirmation.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis to confirm the concentration of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) Ethanol precipitation for PstItest.jpg|thumb|Ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion to confirm there are some PstI cutting site in Ag43(K346007)(cut with EcoRI and SpeI to remove PstI this parts potentially has).<br />
<br />
{|<br />
|Used DNA <br />
|K346007 cut with EcoRI and SpeI<br />
|-<br />
|Concentration(ng/ul)<br />
|25<br />
|-<br />
|Used DNA volume(ul) <br />
|2<br />
|-<br />
|theoretical ez value(ul)<br />
|0.02<br />
|}<br />
<br />
<br />
PstI = 0.1ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.9 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.2 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.2 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.8 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.5 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 1.0 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) cut with PstI(0.1, 0.2, 0.5, 1.jpg|thumb|Digestion result]]<br />
<br />
From this digestion, we confirmed there is at least one PstI cutting site in Ag43 fragment because these digestion results showed same bp and same number of cutting band.<br />
<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm how ligated Ag43 fragment(s) with dT on pSB1AK3.<br />
We used Ag43 last 700bp area as primer which had designed as sequencing primer. '''If three (two forward and one reverse )Ag43 were inserted, this primer can anneal to forward Ag43 as forward primer and reverse Ag43 as reverse primer then amplify about 766bp.''' If there are no reverse Ag43, DNA can't be amplified. <br />
<br /><br /><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
<br />
We failed amplification. <br />
Retry!<br />
<br />
==PCR==<br />
<p><br />
We did PCR written above once again. Change some point of reaction.<br />
We amplified Ag43(K346007) original DNA also as a control.<br /><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
</p><br />
<br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 22nd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Results of digestion at 21st.<br /><br />
We expected to appear the band of about 700bp.<br />
So, we use 1% and 2% agarose gel.<br />
<br />
[[image:HokkaidoU_2012_120722_ag43_pcr-1%_print-1.jpg|thumb|PCR result image]]<br />
[[image:HokkaidoU2012_120722_ag43_pcr-2%_print.jpg|thumb|PCR result image]]<br />
<br />
<br /><br />
We can't find some band in 700~800bp...<br />
</p><br />
<br />
==Liquid Culture==<br />
<p><br />
Liquid culture for single colony isolated Ag43-dT on pSB1AT3.<br />
<br />
#Picked up one colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBA.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
Digested Ag43(K346oo7) plasmid extraction product with EcoRI and SpeI.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:58:34Z
<p>Slecat: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed to incubate in liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br /><br />
Digestion recipe<br /><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br /><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br /><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br /><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br /><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br /><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br /><br />
Ligation recipe<br /><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br /><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br /><br /><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see July 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br /><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br /><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_11
Team:HokkaidoU Japan/Notebook/aggregation Week 11
2012-09-26T19:53:27Z
<p>Slecat: /* Digestion of RBS-phaB-pSB1A2 as Vector and RBS-eYFP-dT as Insert */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 10th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 by EcoRI and SpeI, Ag43-dT-pSB1AK3 (previously digested by HindIII) by XbaI and NotI, and pSTV28 by EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 by XbaI and SpeI as a control for confirmation of the ability of restriction enzyme.<br />
<br /><br />
Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
Insert2 (Ag43-dT-pSB1AK3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|25 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xK buffer<br />
|1.5 ul<br />
|-<br />
|100xBSA<br />
|0.3ul<br />
|-<br />
|DW<br />
|1.5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (15 ng/ul)<br />
|9 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|10xBSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30~40 ng/ul)<br />
|10 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120910 digestion Insert1-pBAD-RBS-eCFP-RBS-pSB1A2 Insert2-Ag43-dT-pSB1AK3 Vector-ptet-RBS-eYFP-dT-pSTV28 Control-pT7-RBS-pSB1A2.jpg|thumb|digestion result]]<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120910 EthaPre Insert1-Insert2-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert1 DNA solution is 50 ng/ul, Insert2 DNA solution is 10~15 ng/ul and Vector DNA solution is 10 ng/ul.<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (10 ng/ul)<br />
|4 ul<br />
|-<br />
|Insert1 DNA (50 ng/ul)<br />
|2 ul<br />
|-<br />
|Insert2 DNA (10~15 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
Transformation of DH5&alpha;.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 14 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 11th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2==<br />
<p><br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
*We noticed that this step was actually 68.9 degree after reaction. <br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 542bp.<br />
<br />
[[image:HokkaidoU2012 120911 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
Because of mis-setting the PCR program, we failed to amplify desired DNA fragment so we retried the colony PCR in same reagent, correct PCR machine program.<br />
<br />
[[image:HokkaidoU2012 120912 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed that the ligated DNA contains at least Ag43-dT-BioBrick_Suffix complex. We selected No. 2,4 for incubation for plasmid extraction and No. 5,6 for Aggregation check.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 12th==<br />
<div><br />
==Analysis nucleotide sequence==<br />
<p><br />
We analyzed the nucleotide sequence of pBAD-RBS-Ag43-dT on pSB1AK3 and pSB1C3.<br />
We used these 6 kinds of primers.<br />
100b up EX-F primer, pBAD-f1 primer, pBAD-f2 primer, Ag43-f1 primer, Ag43-f2 primer, Ag43-f3 primer, Ag43-f4 primer, 200b down PS-R primer<br />
<br />
</p><br />
<br />
==Aggregation check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
Aggregation check for No. 5,6 colonies selected by colony PCR at August 11th.<br />
#Prepared 5 ml LBA into glass tubes.<br />
#Re-suspended 2 colony mixture (No. 2 and No. 5 respectively). <br />
#Incubated at 37C for 18 hrs.<br />
</p><br />
<br />
==Digestion of RBS-phaB-pSB1A2 as Vector and RBS-eYFP-dT as Insert==<br />
<p><br />
To make a construct of RBS-phaB-RBS-eYFP-dT-pSB1A2, we digested RBS-phaB-pSB1A2 with SpeI & PstI, RBS-eYFP-dT with XbaI & PstI.<br />
<br />
<br />
Insert (RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|25 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xK buffer<br />
|1.5 ul<br />
|-<br />
|100xBSA<br />
|0.3 ul<br />
|-<br />
|DW<br />
|1.5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS-phaB-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120912 Digestion Insert(RBS-eYFP-dT)-Vector(RBS-phaB-pSB1A2)-Control(pBAD-RBS-eCFP-RBS-pSB1A2).jpg|thumb|digestion result]]<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120912 Ethapre 20min Insert-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 40 ng/ul and Vector DNA solution is 30 ng/ul.<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (40 ng/ul)<br />
|3 ul<br />
|-<br />
|Insert DNA (30 ng/ul)<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 13th==<br />
<div><br />
==Transformation of RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product in JM109.<br />
#Mixed 2 ul ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 14 hours.<br />
</p><br />
<br />
==Aggregation Check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
#Mixed 25 ul Arabinose solution (20 %) to 5 ml LBC in glass tube.<br />
#Incubated for 24 hrs at 37C.<br />
<br />
<br />
Result: The construct has aggregated not forming large cluster but small dot cluster, and the supernatant has muddiness.<br />
</p><br />
<br />
==Colony PCR of RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R down primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|64.4<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaB-pSB1A2)as controls.<br />
Desired product is about 1800~2000bp.<br />
<br />
[[image:HokkaidoU2012 120913 colop RBS-phaB-RBS-eYFP-dT-pSB1A2 1,2,,,N1,N2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.<br />
</p><br />
<br />
<br />
==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 1 and No. 2 respectively). <br />
#Incubated at 37C for 14 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 14th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.<br />
<br />
Insert (RBS-phaB-RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|21 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(RBS-phaC-RBS-phaA-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120914 Digestion Insert Vector.jpg|thumb|digestion result]]<br />
<br />
We suppose the Insert DNA (RBS-phaB-RBS-eYFP-dT) has contained Vector-dimer DNA as contamination. But desired DNA fragment (about 1600 bp) bond also appeared in the result of migration. We picked up the fragment and extracted from gel.<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying in room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120914 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (50~60 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 13 hours.<br />
<br />
We succeeded to transform the cells but noticed that we have used wrong DNA solution. We'll try this DNA synthesis again tomorrow.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 15th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.<br />
<br />
Insert (RBS-phaB-RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|21 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(RBS-phaC-RBS-phaA-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120915 Digestion Insert-Vector.jpg|thumb|digestion result]]<br />
<br />
</p><br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying in room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120915 Ethapre Insert-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (50~60 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product in JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 17 hours.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 16th==<br />
<div><br />
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pT7-RBS-pSB1C3==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI, and pT7-RBS-pSB1C3 with EcoRI and SpeI. As a control, we digested pT7-RBS-pSB1C3 with only EcoRI to confirm the digestibility of This DNA (The digestibility with SpeI has confirmed in August 26th).<br />
<br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 35ng/ul)<br />
|41 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
Vector(pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg|thumb|digestion result]]<br />
<br />
We confirmed 4 bonds in the result of Inset DNA digestion. A Bond which shows the highest concentration and places about 8k ~ 10k bp area would be pBAD-RBS-eCFP-RBS-Ag43-pSTV28 construct. We considered about it bond whether remained from digestion, or at first formed dimer and separated by digestion. In either case, our target product has about 5200 bp and there is appropriate bond. We extracted the target bond from TBE gel and went next step.<br />
</p><br />
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying in room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA <br />
|4 ul<br />
|-<br />
|Vector DNA<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
Transformation of DH5&alpha;.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 13 hours.<br />
</p><br />
<br />
==Colony PCR of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(phaA-1083bp-F primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R down primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaC-RBS-phaA-RBS-phaB-dT-pSB1A2)as controls.<br />
Desired product is about 1800~2000bp.<br />
<br />
[[image:HokkaidoU2012 120916 coloP RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.<br />
</p><br />
<br />
==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 1 and No. 2 respectively). <br />
#Incubated at 37C for 20 hrs.<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_9
Team:HokkaidoU Japan/Notebook/aggregation Week 9
2012-09-26T19:51:04Z
<p>Slecat: /* Digestion of pSB1AK3 with hindIII */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 27th==<br />
<div><br />
<br />
==Aggregation check==<br />
<p><br />
We found a lot of colonies on LBA plate, spreaded at August 26th.<br />
We incubated 3 colonies in LBAK solution including 1% L-arabinose for 18 hrs.<br />
However, we could not find expression of Ag43 protein.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
</p><br />
[[image:HokkaidoU2012_120827_take-iroiro.jpg|thumb|Plasmid extraction resulsts]]<br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120827 colop pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
The result did not show the band clearly. We selected No.1 and 2 colony for incubation.<br />
<br />
<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium.<br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 2 colonies (No.1 and No.2 respectively). <br />
#Incubated at 37C for 15 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2==<br />
<p><br />
#Mixed 1 ul DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours. <br />
</p><br />
<br />
==PCR of RBS-YFP-dT==<br />
<p><br />
Amplified the construct with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.2<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 28th==<br />
<div><br />
<br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by EcoRI and SpeI.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120828_pbad-rbs-ag43-dt-dig.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
<br />
==Plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3. We re-suspended No.1,2 colonies and incubated.<br />
<br />
[[image:HokkaidoU2012 120828 mini-prep pT7-RBS-Ag43-dT No.jpg|thumb|Plasmid extraction result]]<br />
<br />
</p><br />
<br />
==Estimation of Concentration of RBS-eYFP-dT and ptetR-pSB1A2==<br />
<p><br />
{|<br />
|Solution<br />
|Value (ul)<br />
|-<br />
|1kb ladder<br />
|1<br />
|-<br />
|Insert<br />
|1<br />
|-<br />
|Vector<br />
|1<br />
|-<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120828 Vector-ptetonpSB1C3-Concentrationcheck Insert-rbs-yfp-dt-PCR.jpg|thumb|Electrophoresis result]]<br />
<br />
<br />
From this result, We estimated that the concentration of Insert DNA solution is about 40 ng/ul, and Vector DNA is also about 40 ng/ul.<br />
</p><br />
<br />
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==<br />
<p><br />
ptet-pSB1A2(Vector) was digested with SpeI and PstI, and RBS-Ag43-dT(Insert) was cut with XbaI and PstI.<br />
<br />
<br />
<br />
<br />
Vector<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|5 ul<br />
|-<br />
|SpeI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Inset<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|14 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120828 Digestion Insert-rbs-yfp-dt-PCR(XP) Vector-ptetonpSB1C3(SP).jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that Insert and Vector DNA were digested.<br />
</p><br />
<br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for ptet-pSB1A2 and RBS-eYFP-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW. <br />
</p><br />
<br />
<br />
==Ligation==<br />
<p><br />
Ligation for ptet-pSB1A2 and RBS-eYFP-dT.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|3 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product (ptet-RBs-eYFP-dT on pSB1A2) in DH5&alpha;.<br />
#Mixed 1 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LBA.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hrs.<br />
</p><br />
<br />
<br />
==Gel extraction product check of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
We couldn't get desired plasmid DNA by transformation. We doubted contamination of non-digested vector DNA and decided to test the gel extraction product of pT7-RBS on pSB1C3 was successfully separated from non-digested product or not by transformation.<br />
#Mixed 1 ul of each DNA of ligation product and digestion product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 16 hours. <br />
<br />
<br />
There were no colony on the LBC plate that was spread solution mixed digestion product.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 29th==<br />
<div><br />
==Aggregation check==<br />
<p><br />
We incubated 9 colonies transformed at August 25th in LBAK solution including 1% L-arabinose for 16 hrs.<br />
Then we find that 1 culture expressing Ag43.<br />
Observed the E. coli cluster by a microscope of 100 magnifications.<br />
<br />
<br />
Then we checked whether pBAD promoter is behaving exactly or not.<br />
#1 colony ( could expressing Ag43 ) incubated in 2 ml LB at 37C for 2 hrs.<br />
#Prepared 5 kinds of LB medium differing the concentration of L-arabinose.<br />
<br />No. 1 :0.001%<br />
<br />No. 2 :0.1% <br />
<br />No. 3 :0.4%<br />
<br />No. 4 :1.0%<br />
<br />No. 5 :0%<br /><br />
#Added 400 ul of culture in several LB medium.<br />
#Incubated for 17 hrs and 30 min at 37C.<br />
<br />
</p><br />
<br />
==PCR of eCFP(E0020)==<br />
<p><br />
Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Desired product is about 800~900bp.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.2<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR of pT7-RBS-Ag43-dT on pSB1C3 transformed at August 28th. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp. This length is almost same as N2.<br />
<br />
[[image:HokkaidoU2012 120829 colop pT7-RBs-Ag43-dTonpSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We decided to incubate the No.7 and 8 colony.<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of pT7-RBS-Ag43-dTonpSB1C3 and Ag43-dTonpSB1AK3==<br />
<p><br />
Incubation of pT7-RBS-Ag43-dTonpSB1C3 (and Ag43-dT on pSB1AK3) in LBC (LBA) liquid medium.<br />
#Prepared 2 ml LBC (LBA) into culture tubes.<br />
#Re-suspended 2 colonies No.7 and No.8 respectively. Ag43-dT on pSB1AK3 was re-suspended the N2 colony. <br />
#Incubated at 37C for 17 hrs.<br />
</p><br />
<br />
<br />
==Estimation of Concentration of RBS-eYFP-dT (PCR product) and ptetR-pSB1A2==<br />
<p><br />
{|<br />
|Solution<br />
|Value (ul)<br />
|-<br />
|1kb ladder<br />
|1<br />
|-<br />
|Insert<br />
|1<br />
|-<br />
|Vector<br />
|1<br />
|-<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120829 PCR 100bpup-eCFP-200bpdown mini-prep B0034.jpg|thumb|Electrophoresis result]]<br />
<br />
<br />
From this result, We estimated that the concentration of Insert DNA solution is about 36 ng/ul, and Vector DNA is also about 29 ng/ul.<br />
</p><br />
<br />
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==<br />
<p><br />
ptet-pSB1A2(Vector) was digested with EcoRI and SpeI, and RBS-Ag43-dT(Insert) was cut with EcoRI and XbaI.<br />
And we used construct of pT7-RBS on pSB1C3 as control for the function of XbaI.<br />
<br />
<br />
<br />
Insert (eCFP)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (36 ng/ul)<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS on pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (29 ng/ul)<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBs on pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|100x BSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120829 Digestion eCFP RBSonpSB1A2 XbaIcontrol.jpg|thumb|digestion result]]<br />
<br />
From this result, Vector DNA were digested by XbaI, but remains some amount of non-digested DNA.<br />
And insert DNA solution were not existed in both d- and d+ lines.It means we failed to extract from gel after electrophoresis. We decided to retry PCR.<br />
</p><br />
<br />
==PCR of eCFP(E0020)==<br />
<p><br />
Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Desired product is about 800~900bp.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.0<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 30th==<br />
<div><br />
<br />
==Estimation of concentration of eCFP, pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT-pSB1AK3==<br />
<p><br />
No. 7,8 means the colony number of colony PCR of pT7-RBS-Ag43-dT-pSB1C3.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP mini-prep pT7-RBS-Ag43-dTonpSB1C3 No.jpg|thumb|electrophoresis result]]<br />
<br />
<br />
We failed the PCR for E0020.<br />
We estimated the concentration of pT7-RBS-Ag43-dT-pSB1C3 is 31 ng/ul and Ag43-dT-pSB1AK3 is 35 ng/ul.<br />
</p><br />
<br />
==Digestion of pT7-RBS-pSB1C3 and Ag43-dT-pSB1AK3==<br />
<p><br />
Digestion of pT7-RBS-pSB1C3 (as Vector) and Ag43-dT-pSB1AK3 (as Insert) to make pT7-RBs-Ag43-dT-pSB1C3 construct.<br />
<br />
Vector<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (31 ng/ul)<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xm buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Inset<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|14 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
As a background, we did PCR for eCFP again.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP digestion-Ag43-dTonpSB1AK3.jpg|thumb|digestion and PCR result]]<br />
<br />
<br />
We did not do digestion of vector DNA sokution, result, because it has only too low concentration to be digested product solution.<br />
<br />
<br />
We failed PCR again.<br />
The digestion result was confirmed that the Insert DNA was successfully digested by XbaI and SpeI.<br />
And we extracted the insert DNA digestion product from agarose gel by gel-extraction kit and got 20 ul of Insert DNA solution. <br />
</p><br />
<br />
==Digestion of pSB1AK3 by hindIII==<br />
<p><br />
The gel-extracted DNA solution contains Ag43-dT DNA and pSB1AK3 DNA because these DNA have same size of base pair and exist as same band in agarose gel. Thus we digest pSB1AK3 with HindIII to separate it from Ag43-dT.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|20 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|10<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
As a background, we did PCR for eCFP again.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP digestion(HindIII)-Ag43-dTonpSB1AK3.jpg|thumb|PCR and digestion result]]<br />
<br />
<br />
We failed the PCR for E0020.<br />
The digestion result was confirmed that the Insert DNA was successfully digested with HindIII.<br />
</p><br />
<br />
==Ethanol precipitation of pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT==<br />
<p><br />
We did ethanol precipitation to get more high concentration DNA solution of each part. And we got 5ul of each DNA solution.<br />
<br />
[[image:HokkaidoU2012 120831 Insert-d--d+ Vector-d+.jpg|thumb|ethanol precipitation result]]<br />
<br />
The electrophoresis result showed that the pT7-RBS-pSB1C3 construct was successfully digested by SpeI and get enough concentration of DNA solution to ligate. <br />
</p><br />
<br />
==Ligation of pT7-RBS-pSB1C3 and Ag43-dT==<br />
<p><br />
Ligation and pT7-RBS-pSB1C3 (as Vector) and Ag43-dT (as Insert).<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|2.5 ul<br />
|-<br />
|Insert DNA<br />
|2.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
[[image:HokkaidoU2012 120831 Ligation Vector Insert Ligationproduct(4ul).jpg|thumb|ligation result]]<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of Ag43-dT-pSB1AK3 and ptet-RBS-YFP-dT-pSB1A2==<br />
<p><br />
Incubation of Ag43-dT-pSB1AK3 and ptet-RBS-YFP-dT-pSB1A2 in LBA liquid medium.<br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 1 colony from each plate that each construct plated. <br />
#Incubated at 37C for 22 hrs (Ag43-dT-pSB1AK3 was Incubated for 41 hrs)<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 31st==<br />
<div><br />
==Plasmid extraction for pBAD-RBS-Ag43-dT on pSB1AK3==<br />
<p><br />
We used promega SV miniprep kit.<br />
We got 100 ul DNA solution.<br />
</p><br />
[[image:HokkaidoU2012_120831_pbad-rbs-ag43-dt-on1ak3.jpg|thumb|plasmid extraction result]]<br />
<br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by EcoRI and SpeI.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120831_take-iroiro2.jpg|thumb|digestion result]]<br />
<br />
==Transformation of pT7-RBS-Ag43-dT-pSB1C3 and eCFP (E0020)==<br />
<p><br />
#Mixed 2 ul ligation product and 1ul E0020 DNA solution to each 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA and LBC).<br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol (E0020 mixture skipped this step).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours.<br />
<br />
We failed to transformation of pT7-RBS-Ag43-dT-pSB1C3. <br />
</p><br />
<br />
==Confirmation of amplification of ptet-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
After plasmid extraction, we confirmed whether the desired construct ptet-RBS-eYFP-dT was really amplified or not by PCR.<br />
We chose EX-F primer as forward primer and PS-R primer as reverse primer then amplified about 1000bp of insert DNA.<br />
As controls, we chose DW as N1 and ptet-RBS-eCFP-dT construct as N2.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-F primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
[[image:HokkaidoU2012 120831 colop N1(DW) N2(ptet-RBS-eCFP-dT) 1(ptet-RBS-eYFP-dT).jpg|thumb|PCR result]]<br />
<br />
<br />
This image showed that the desired construct (about 1000 bp) amplified by incubation.<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of eCFP-pSB1A2==<br />
<p><br />
Incubation of eCFP-pSB1A2 in LBA liquid medium.<br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 1 colony. <br />
#Incubated at 37C for 16 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS-eYFP-dT-pSB1A2 and pT7-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
#Mixed 2 ul pT7-RBS-Ag43-dT-pSB1C3 ligation product and 1ul ptet-RBS-eYFP-dT-pSB1A2 DNA solution to each 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA and LBC).<br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol (ptet-RBS-eYFP-dT-pSB1A2 mixture skipped this step).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 1st==<br />
<div><br />
==Digestion for pSB1C3==<br />
<p><br />
We have to change the plasmid backbone to pSB1C3.<br />
We got the insert DNA at August 31th.<br />
So we need to get the pSB1C3 solution digested by EcoRI and SpeI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|3 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120902_take-iroiro.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of pBAD-RBS-Ag43-dT solution and pSB1C3 solution digested by EcoRI & SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for insert DNA and pSB1C3.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|1 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 1 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Plated 300 ul of the culture onto first LBC dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBC dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
</p><br />
<br />
==Plasmid extraction of Ag43-dT-pSB1AK3 and RBS-pSB1A2==<br />
<p><br />
We used plasmid extraction kit of Nippon genetics and got 50 ul Ag43-dT-pSB1AK3 plasmid DNA solution.<br />
</p><br />
<br />
==Digestion of eCFP-pSB1A2 and RBS-pSB1A2==<br />
<p><br />
To make a construct of eCFP-RBS-pSB1A2, we digested eCFP-pSB1A2 by EcoRI and SpeI, and RBS-pSB1A2 with EcoRI and XbaI. And we digested pT7-RBS-pSB1C3 by XbaI as a control for confirmation of the ability to digest.<br />
<br />
Insert (eCFP-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|11 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (29 ng/ul)<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|100x BSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120902 Digestion I-+ V-+ C(XbaI)-+.jpg|thumb|digestion result]]<br />
<br />
<br />
From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 2nd==<br />
<div><br />
==Ethanol precipitation of digestion products (eCFP and RBS-pSB1A2) and estimation of concentration==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120902 Ethapre V I.jpg|thumb|ethanol precipitation result]]<br />
<br />
We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 37 ng/ul.<br />
</p><br />
<br />
==Incubation of pT7-RBS-Ag43-dT-pSB1C3 transformation product==<br />
<p><br />
We failed to cultivation of above construct two times so we tried to incubation in LBC liquid medium.<br />
#Prepared 1.8 ml LBC into culture tubes.<br />
#Re-suspended 200 ul transformation product solution (which was mixed with LB and incubated for 3 hrs). <br />
#Incubated at 37C. <br />
</p><br />
<br />
==Ligation of eCFP and RBS-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (37 ng/ul)<br />
|1 ul<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|11 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of eCFP-RBS-pSB1A2==<br />
<p><br />
#Mixed 2 ul eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 19 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<br />
<div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_8
Team:HokkaidoU Japan/Notebook/aggregation Week 8
2012-09-26T19:48:13Z
<p>Slecat: /* liquid culture */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 20th==<br />
<div><br />
==Single colony isolation==<br />
<p><br />
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.<br />
#Picked up one colony.<br />
#Incubated on LBK (dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp up primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls.<br />
Desired product is about 300~400bp.<br />
<br />
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.<br />
</p><br />
<br />
<br />
==PCR==<br />
<p><br />
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Liquid culture==<br />
<p><br />
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBK (LBC) into culture tubes.<br />
#Resuspended 1 colonies. <br />
#Incubated the tubes at 37C for 16 hours (19 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 21st==<br />
<div><br />
==PCR==<br />
<p><br />
PCR of pBAD(containing araC)-RBS.<br />
And, we checked the plasmid which we refined by plasmid extraction at August 18th is pBAD-RBS on pSB1A3 or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-rbs_pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Aggregation check==<br />
<p><br />
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK.<br />
We checked the construct by induction of L-arabinose after 16 hours incubate.<br />
<br />
#2 ml of liquid culture divided two culture. (made two 1 ml culture)<br />
#Added 1 ml LBK in one culture as a negative control.<br />
#Added 900 ul LBK and 100 ul 20% L-arabinose.<br />
#Incubated at 37C 130 rpm for 2 hrs and 30 min.<br />
#Placed tubes on the table at 30 min.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used Plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
<br />
[[image:HokkaidoU2012 120821 pT7-RBS on pSB1C3 ColonyNo. 4,5 mini-prep.jpg|thumb|Plasmid extraction result]]<br />
<br />
<br />
The concentration of 20 ul of plasmid extraction products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2. <br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBA (LBC) into culture tubes.<br />
#Resuspended 2 colonies. <br />
#Incubated the tubes at 37C for 18 hours (16 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 22nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|plasmid extraction result]]<br />
<br />
<br />
<br />
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.<br />
We tried plasmid extraction to the latter, we got the 20 ul of DNA solution.<br />
And then, we did electrophoresis the plasmid extraction products and (pBAD-RBS and pBAD) gel extract products.<br />
<br />
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]<br />
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR of pT7-RBS on pSB1C3.<br /><br />
<br />
We used 4 kinds of primer set.<br />
<br /><br />
1 : EX-F , PS-R primer<br /><br />
2 : EX-F , 200b down primer<br /><br />
3 : 100b up , PS-R primer<br /><br />
4 : 100b up , 200b down primer<br /><br />
The density of primer solutions is 10 uM.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer<br />
|1 ul<br />
|-<br />
|Reverse Primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 23rd==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3.<br />
<br />
Digestion of pBAD-RBS.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pBAD-RBS (100 ng/ul)<br />
|12 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Digestion of Ag43-dT on pSB1AK3.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT (120 ng/ul)<br />
|7 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120824_ag43-dt-dig2.jpg|thumb|Ag43-dT digestion result]]<br />
[[image:HokkaidoU2012_120824_pbad-rbs-dig.jpg|thumb|pBAD-RBS digestion result]]<br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
</p><br />
==Digestion==<br />
<p><br />
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 257ng/ul)<br />
|9 ul<br />
|-<br />
|HindIII(15 U/ul)<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]<br />
<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained as non-digested products.<br />
<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 24th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.<br />
<br /><br />
<br /><br />
Ag43-dT on pSB1AK3<br />
<br /><br /><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pBAD-RBS<br /><br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 20ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]<br />
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.<br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|4 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 25th==<br />
<div><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and electrophoresis result image of ligation products, then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligated DNA solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. <br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|600 (10 hours)<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 26th==<br />
<div><br />
<br />
===Ligation===<br />
<p><br />
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS<br />
|3 ul<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Plated 300 ul of the culture onto first LBA dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.<br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|0.25 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|DW<br />
|1.75 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_8
Team:HokkaidoU Japan/Notebook/aggregation Week 8
2012-09-26T19:47:33Z
<p>Slecat: /* liquid culture */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 20th==<br />
<div><br />
==Single colony isolation==<br />
<p><br />
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.<br />
#Picked up one colony.<br />
#Incubated on LBK (dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp up primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls.<br />
Desired product is about 300~400bp.<br />
<br />
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.<br />
</p><br />
<br />
<br />
==PCR==<br />
<p><br />
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Liquid culture==<br />
<p><br />
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBK (LBC) into culture tubes.<br />
#Resuspended 1 colonies. <br />
#Incubated the tubes at 37C for 16 hours (19 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 21st==<br />
<div><br />
==PCR==<br />
<p><br />
PCR of pBAD(containing araC)-RBS.<br />
And, we checked the plasmid which we refined by plasmid extraction at August 18th is pBAD-RBS on pSB1A3 or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-rbs_pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Aggregation check==<br />
<p><br />
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK.<br />
We checked the construct by induction of L-arabinose after 16 hours incubate.<br />
<br />
#2 ml of liquid culture divided two culture. (made two 1 ml culture)<br />
#Added 1 ml LBK in one culture as a negative control.<br />
#Added 900 ul LBK and 100 ul 20% L-arabinose.<br />
#Incubated at 37C 130 rpm for 2 hrs and 30 min.<br />
#Placed tubes on the table at 30 min.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used Plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
<br />
[[image:HokkaidoU2012 120821 pT7-RBS on pSB1C3 ColonyNo. 4,5 mini-prep.jpg|thumb|Plasmid extraction result]]<br />
<br />
<br />
The concentration of 20 ul of plasmid extraction products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2. <br />
<br />
</p><br />
<br />
==liquid culture==<br />
<p><br />
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBA (LBC) into culture tubes.<br />
#Resuspended 2 colonies. <br />
#Incubated the tubes at 37C for 18 hours (16 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 22nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|plasmid extraction result]]<br />
<br />
<br />
<br />
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.<br />
We tried plasmid extraction to the latter, we got the 20 ul of DNA solution.<br />
And then, we did electrophoresis the plasmid extraction products and (pBAD-RBS and pBAD) gel extract products.<br />
<br />
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]<br />
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR of pT7-RBS on pSB1C3.<br /><br />
<br />
We used 4 kinds of primer set.<br />
<br /><br />
1 : EX-F , PS-R primer<br /><br />
2 : EX-F , 200b down primer<br /><br />
3 : 100b up , PS-R primer<br /><br />
4 : 100b up , 200b down primer<br /><br />
The density of primer solutions is 10 uM.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer<br />
|1 ul<br />
|-<br />
|Reverse Primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 23rd==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3.<br />
<br />
Digestion of pBAD-RBS.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pBAD-RBS (100 ng/ul)<br />
|12 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Digestion of Ag43-dT on pSB1AK3.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT (120 ng/ul)<br />
|7 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120824_ag43-dt-dig2.jpg|thumb|Ag43-dT digestion result]]<br />
[[image:HokkaidoU2012_120824_pbad-rbs-dig.jpg|thumb|pBAD-RBS digestion result]]<br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
</p><br />
==Digestion==<br />
<p><br />
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 257ng/ul)<br />
|9 ul<br />
|-<br />
|HindIII(15 U/ul)<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]<br />
<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained as non-digested products.<br />
<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 24th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.<br />
<br /><br />
<br /><br />
Ag43-dT on pSB1AK3<br />
<br /><br /><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pBAD-RBS<br /><br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 20ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]<br />
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.<br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|4 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 25th==<br />
<div><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and electrophoresis result image of ligation products, then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligated DNA solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. <br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|600 (10 hours)<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 26th==<br />
<div><br />
<br />
===Ligation===<br />
<p><br />
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS<br />
|3 ul<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Plated 300 ul of the culture onto first LBA dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.<br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|0.25 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|DW<br />
|1.75 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_8
Team:HokkaidoU Japan/Notebook/aggregation Week 8
2012-09-26T19:46:44Z
<p>Slecat: /* Single colony isolation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 20th==<br />
<div><br />
==Single colony isolation==<br />
<p><br />
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.<br />
#Picked up one colony.<br />
#Incubated on LBK (dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp up primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls.<br />
Desired product is about 300~400bp.<br />
<br />
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.<br />
</p><br />
<br />
<br />
==PCR==<br />
<p><br />
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==liquid culture==<br />
<p><br />
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBK (LBC) into culture tubes.<br />
#Resuspended 1 colonies. <br />
#Incubated the tubes at 37C for 16 hours (19 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 21st==<br />
<div><br />
==PCR==<br />
<p><br />
PCR of pBAD(containing araC)-RBS.<br />
And, we checked the plasmid which we refined by plasmid extraction at August 18th is pBAD-RBS on pSB1A3 or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-rbs_pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Aggregation check==<br />
<p><br />
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK.<br />
We checked the construct by induction of L-arabinose after 16 hours incubate.<br />
<br />
#2 ml of liquid culture divided two culture. (made two 1 ml culture)<br />
#Added 1 ml LBK in one culture as a negative control.<br />
#Added 900 ul LBK and 100 ul 20% L-arabinose.<br />
#Incubated at 37C 130 rpm for 2 hrs and 30 min.<br />
#Placed tubes on the table at 30 min.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used Plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
<br />
[[image:HokkaidoU2012 120821 pT7-RBS on pSB1C3 ColonyNo. 4,5 mini-prep.jpg|thumb|Plasmid extraction result]]<br />
<br />
<br />
The concentration of 20 ul of plasmid extraction products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2. <br />
<br />
</p><br />
<br />
==liquid culture==<br />
<p><br />
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBA (LBC) into culture tubes.<br />
#Resuspended 2 colonies. <br />
#Incubated the tubes at 37C for 18 hours (16 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 22nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|plasmid extraction result]]<br />
<br />
<br />
<br />
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.<br />
We tried plasmid extraction to the latter, we got the 20 ul of DNA solution.<br />
And then, we did electrophoresis the plasmid extraction products and (pBAD-RBS and pBAD) gel extract products.<br />
<br />
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]<br />
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR of pT7-RBS on pSB1C3.<br /><br />
<br />
We used 4 kinds of primer set.<br />
<br /><br />
1 : EX-F , PS-R primer<br /><br />
2 : EX-F , 200b down primer<br /><br />
3 : 100b up , PS-R primer<br /><br />
4 : 100b up , 200b down primer<br /><br />
The density of primer solutions is 10 uM.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer<br />
|1 ul<br />
|-<br />
|Reverse Primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 23rd==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3.<br />
<br />
Digestion of pBAD-RBS.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pBAD-RBS (100 ng/ul)<br />
|12 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Digestion of Ag43-dT on pSB1AK3.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT (120 ng/ul)<br />
|7 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120824_ag43-dt-dig2.jpg|thumb|Ag43-dT digestion result]]<br />
[[image:HokkaidoU2012_120824_pbad-rbs-dig.jpg|thumb|pBAD-RBS digestion result]]<br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
</p><br />
==Digestion==<br />
<p><br />
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 257ng/ul)<br />
|9 ul<br />
|-<br />
|HindIII(15 U/ul)<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]<br />
<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained as non-digested products.<br />
<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 24th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.<br />
<br />
<br />
Ag43-dT on pSB1AK3<br />
<br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pBAD-RBS<br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 20ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]<br />
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.<br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|4 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 25th==<br />
<div><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and ligation products electrophoresis result image then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligation reaction solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. <br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|600 (10 hours)<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 26th==<br />
<div><br />
<br />
===Ligation===<br />
<p><br />
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS<br />
|3 ul<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Plated 300 ul of the culture onto first LBA dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.<br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|0.25 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|DW<br />
|1.75 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
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{{Team:HokkaidoU_Japan/footer}}</div>
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http://2012.igem.org/Team:HokkaidoU_Japan/Notebook
Team:HokkaidoU Japan/Notebook
2012-09-26T19:45:15Z
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Team:HokkaidoU Japan/Notebook/aggregation Week 7
2012-09-26T19:40:43Z
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<div class="hokkaidou-notebook-daily"><br />
==August 16th==<br />
<div><br />
==ligation==<br />
<p><br />
We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS (5 ng/ul)<br />
|1 ul<br />
|-<br />
|Ag43-dT (25 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Ag43-dT on pSB1AK3 digestion with SpeI and XbaI.<br />
<br />
Ag43-dT<br />
SpeI and XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
[[image:HokaidoU2012_120813_pbad-rbs-ori-x10.jpg|thumb|pBad-RBS on pSB1A3 electrophoresis result]]<br />
[[image:HokkaidoU2012_120813_ag43-dt-s-x.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 17th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1 colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls.<br />
Desired product is about 800~1000bp.<br />
<br />
[[image:HokkaidoU2012_120817_pbad-rbs-ag43-dt-coloP.jpg|thumb|Colony PCR result]]<br />
<br />
From this result, N2 has about 500bp band.<br />
We use to liquid culture, No. 7,10,12.<br /><br />
We cultured these DNA in E. coli solution, after added 200 ul LB, at 37C for 3 hrs.<br />
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for hours at 37C.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of BBa_I13453 (pBAD on pSB1A3) in DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 19 hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
From sequencing results, we found that we failed to make pT7-RBS on pSB1K3.<br /><br />
So, we tried to make it again. <br /><br />
When the restriction enzyme digest the DNA, it is important for 3A assembry to be digested the target plasmid completely. So, we activated the restriction enzymes for 10 hours.<br /><br />
<br /><br />
pT7 (BBa_I719005)<br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
RBS (BBa_B0034)<br />
XbaI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
pSB1C3<br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (25 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 18th==<br />
<div><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for three digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Before digestion of pBAD-RBS on pSB1A3 and Ag43-dT on pSB1AK3, it is necessary to refine these two DNA. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 20 ul of DNA solution from 10 ul of plasmid extraction products.<br />
</p><br />
==Digestion==<br />
<p><br />
Digested pBAD-RBS on pSB1A3 as insert and Ag43-dT on pSB1AK3 as vector with EcoRI and SpeI to make a constract, pBAD-RBS-Ag43-dT on pSB1AK3.<br><br />
<br><br />
<br />
pBAD-RBS<br><br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|plasmid extraction product (20 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT on pSB1AK3<br><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Gel extraction product (40 ng/ul)<br />
|8 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|17 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012_120818_pbad-rbs--ag43-dt-dig.jpg|thumb|Digestion result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for colony number of No. 10 and 12 of pBAD-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
[[image:HokkaidoU2012_120818_pbad-rbs-ag43-dt.jpg|thumb|plasmid extraction result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for pT7 and RBS as inserts and pSB1C3 as vector.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7 (100 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT (100 ng/ul)<br />
|2 ul<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS (80 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT on pSB1AK3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 19th==<br />
<div><br />
==Transformation==<br />
<p><br />
Transformation of ligation product (two construct ligated at August 18) in DH5&alpha;.<br /><br />
<br />
<br />
pT7-RBS on pSB1C3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBC.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 18 hours.<br />
<br />
pBAD-RBS-Ag43-dT on pSB1AK3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LBK.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 16 hours and 30 minutes.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR for pT7-RBS on pSB1K3.<br />
By sequencing reaction, we found the No. 10 plasmid is not pT7-RBS on pSB1K3.<br />
We checked the other plasmid is the same or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-Forward primer : 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-Reverse primer : 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|30<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 45<br />
<br />
[[image:HokkaidoU2012_120819_pcr.jpg|thumb|PCR result]]<br />
<br />
No. 4 plasmid has independent bands.<br />
However, if it is pT7-RBS on pSB1K3, it has about 60~70bp band.<br />
<br />
</p><br />
<br />
</div><div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_5
Team:HokkaidoU Japan/Notebook/aggregation Week 5
2012-09-26T19:33:04Z
<p>Slecat: /* digestion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 30th==<br />
<div><br />
==Digestion==<br />
<p><br />
I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.<br />
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
I think that the E. coli which we used transformation is BL21(DE3)pLysS.<br />
So, the E. coli could grow on LBC plate.<br /><br />
I'm going to do transformation , use DH5&alpha;.<br />
<br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 200 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish. <br />
#Incubated the plates at 37C for 14 hrs.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 31st==<br />
<div><br />
==Liquid culture==<br />
<p><br />
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 1st==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT which had been incubated from 31st July. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012 120801 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br />
<br><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''pT7-RBS(30 ng/ul) '''=No. 10<br />
<br><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012 120801 pt7-rbs-digS.jpg|thumb|digestion result]]<br />
<br />
We couldn't digested them exactly, so we tried to digest once more time.<br><br />
<br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br><br />
SpeI 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for pBAD-RBS on pSB1K3.<br />
#Added 2 ml of LBK into culture tube.<br />
#Scraped the surface of glycerol stock of construct.<br />
#Incubated the tube at 33C.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 2nd==<br />
<div><br />
<p><br />
==Preparing chemical competent cell==<br />
Preparing chemical competent cell of BL21, JM109 and DH5&alpha;.<br />
Chemical competent cell made in each E. coli strains.<br />
<br><br><br />
Our competent cell Protocol<br />
#Did single colony isolation on LB plate.<br />
#Incubated the plate for 15-19 hours at 37C.<br />
#Lifted colony of E. coli into 2 ml LB.<br />
#Incubated at 37C for 12-16 hrs at 180-200 rpm.<br />
#Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.<br />
#Incubated cells at 20C (over 24 hrs) at 140 rpm.<br />
#Selected culture by measuring OD 600.<br />
#Incubated the 300 ml flask for 10 min on ice.<br />
#Transfered the culture into two 50 ml Falcon tubes.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)<br />
#Collected them to one tube.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 3.2 ml of TB.<br />
#Instilled 0.24 ml of DMSO in precipitant.<br />
#Incubated the 50 ml Falcon tube for 10 min on ice.<br />
#Divided 50 ul of solutions in each 0.5 ml tubes.<br />
#Freezed the suspension by liquid nitrogen.<br />
#Stored at –80C.<br />
<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)<br />
<br />
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|Electrophoresis result]]<br />
There were low concentration band above thick band. This thick band was same as digestion minus band.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 3rd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for the result of digestion.<br />
[[image:HokkaidoU2012 120803 pt7-rbs-dspe1.jpg|thumb|Electrophoresis result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Spread 300 ul of the transformation onto first dish.<br />
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish. <br />
#Incubated the plates at 37C for 15 hrs 45 min.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm Ag43-f4 primer.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-f4 primer<br />
|1 ul<br />
|-<br />
|PS-R primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120803 Ag43 PCR(Forward=ag43-f4 Reverse=PS-R).jpg|thumb|PCR result]]<br />
<br />
From this result, we could see that the ag43-f4 primer had worked and DNA of last 500~600 bp of ag43 to BioBrick suffix were increased.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 4th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012 120804 pt7-rbs-ag43-dt coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band.<br />
Next step, we resuspended these three colonies and incubated. <br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture of colonies passed the colony PCR test.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended 3 colonies.<br />
#Incubated the tubes at 37C for 13 hrs. <br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 5th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
[[image:HokkaidoU2012 120805 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.<br />
<br />
<br />
pT7-RBS on pSB1K3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS (once digestioned with SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 pT7-RBS digestion SpeI (no.jpg|thumb|digestion result]]<br />
From this result, the bottom band was digested incorrectly and middle band was digested correctly, we thought. Was the above band something contaminated in the DNA solution?<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction of middle band. We thought this band would be the exactly digested DNA fragment. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
==Sequencing==<br />
<p><br />
Sequencing to confirm what kind of DNA we made.<br />
<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To extract the PCR product, we did ethanol precipitation.<br />
<br><br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di. <br />
<br />
<br />
Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)<br><br />
We could not get the sequencing data.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 Ag43-dT on pSB1AK3(d+ with XbaI &amp; SpeI) digestion with HindIII.jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_3
Team:HokkaidoU Japan/Notebook/aggregation Week 3
2012-09-26T19:30:09Z
<p>Slecat: /* Ligation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 16th==<br />
<div><br />
Ag43, dT<br />
===Electrophoresis===<br />
<p><br />
Results of digestion at 15th.<br />
<br />
[[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&amp;P-1.jpg|thumb|Digestion result image]]<br />
Product:Ag43(K346007)=3120bp and 2070bp<br />pT7-RBS on pSB1K3=2247bp<br />
<br /><br />
We confirmed there are some kinds of restriction enzyme site in K346007 (digested with SpeI, PstI), and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.<br />
</P><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. Used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution.<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digested and extracted products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW.<br />
</p><br />
<br />
<br><br />
--------<br />
<br><br />
<br />
dT(B0015) would be amplified incorrectly and we couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.<br />
<br />
===PCR===<br />
<p><br />
PCR for dT(B0015)<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp_up forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(200bp_down Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]<br />
<br />
We migrated B0015 of plasmid extraction product, digestion product, and PCR product.<br />
dT done PCR would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
'''Digestion for dT which amplified with PCR.'''<br />
Digested with XbaI and PstI.<br />
<br /><br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
----<br />
<br />
Ag43<br />
<br />
'''Digestion result of Ag43 was incorrect'''. We digested Ag43 once more time.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for Ag43 with SpeI and PstI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43 DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120716 Ag43d- Ag43d+.jpg|thumb|Digestion result image]]<br />
<br />
There are same results with digestion result of recent. '''We thought PstI would cut different site'''.<br />
What is this 500bp fragment????<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50ul of DNA solution.<br />
</p><br />
<br />
----<br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.<br />
<br />
'''Colonies which have Ag43-dT on pSB1T3 were closely existed so we would picked up two or more colonies.''' <br />
#Picked up one (or tow?) colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBC and LBT.<br />
#Incuvated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 17th==<br />
<div><br />
<p><br />
Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3<br />
</p><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products incuvated from yesterday(16th).<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And plasmid extraction products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated.<br />
<br />
<br />
If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp.<br />
And if ligation and transformation, plasmid extraction were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp. <br />
<br />
[[image:HokkaidoU2012 120717 Ag43-Ag43d+(E&amp;S)-(Ag43-dT on pSB1T3)-(pT7-RBS on pSB1C3).jpg|thumb|Digestion and plasmid extraction result image]]<br />
<br />
From this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. '''would PstI not work correctly?''' <br />
<br />
<br />
'''We planed to use dT as vector and Ag43 as Insert.''' A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.<br />
<br />
<br />
About plasmid extraction products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought '''which means we failed single colony isolation then resuspended two another E.coli colonies''' another ligated DNA were transformed. <br />
In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.<br />
<br />
<br />
To confirm plasmid extraction products were really ligated correct DNA fragments, first we extracted Ag43-dT on pSB1T3(low bp band) from TBE gel and digested with EcoRI and PstI.<br />
</p><br />
<br />
----<br />
'''dT Vector plasmid change'''<br />
To use dT as a vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.<br />
<br />
==Digestion==<br />
<p><br />
Digestion to change the plasmid backbone.<br />
Used DNA solution as PCR product(did at 16th) and digestioned pSB1T3 were already exist.<br><br />
<br><br />
<br />
Digestion mix<br><br><br />
<br />
<br />
double digestion(EcoRI and PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717-dt-digestion-print.jpg|thumb|Digestion results]]<br />
<br />
We couldn't confirm digestion results.<br />
<br />
<br />
</p><br />
<br />
----<br />
pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3<br />
<br />
==Electrophoresis and gel extraction==<br />
<p><br />
[[image:HokkaidoU2012 120717-ag43-dt-psb1t3.jpg|thumb|plasmid extraction result]]<br />
'''Gel extraction for Ag43-dT on pSB1T3.'''<br><br />
We cut low bp band (see image below).<br />
Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not.<br />
<br><br><br />
<br />
'''Ag43-dT on pSB1T3(30 ng/ul)'''<br />
<br><br><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br><br />
'''pT7-RBS on pSB1C3(40 ng/ul)'''<br />
<br><br><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717 Ag43 dT(d-,d+E,d+P.jpg|thumb|Digestion result]]<br />
<br />
From this digestion results, <br />
*Cut with EcoRI,PstI and E&P(EcoRI and PstI) showed different base pair bands compared with d-(digestion minus).<br />
*Cut with E&P showed two DNA bands: about 2000bp and 500~1000 bp.<br />
*All of digestion products existed lower place than correct band.Correct DNA would show about 5000bp(EcoRI and PstI), 3000bp and 2000bp (E&P). <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 18th==<br />
<div><br />
<br />
<br />
'''We decided change plasmid backbone of dT pSB1AK3 to pSB1T3.'''<br><br />
If we cut Ag43-dT on pSB1AK3 with EcoRI and PstI to confirm insert DNA, DNA fragment base pair resemble each other(about 3000bp), so then we can't identify them.<br />
<br />
===digestion===<br />
<p><br />
<br />
'''Digestion to confirm what kind of restriction enzyme sites Ag43(K346007) has.'''<br />
<br><br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|- <br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120718-k346007-digestion print.jpg|thumb|Digestion result]]<br />
From this result, we doubted '''are there one or more another PstI resutriction enzyme site except BioBrick suffix'''?<br />
</p><br />
<br />
===PCR===<br />
<p><br />
PCR to confirm what DNA fragment is ligated to vector as insert.<br />
We used PCR for pT7-RBS on pSB1C3. So the result would be 80~90bp band.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Biobrick prefix forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(Biobrick suffix Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
[[image:HokkaidoU2012 120718 pT7-RBS on pSB1C3 EX-F PS-R PCR.jpg|thumb|PCR product]]<br />
</p><br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated(19hrs incubated) Ag43-dT on pSB1T3.<br />
<br />
#Prepared 2 ml LB.<br />
#Added Tet.<br />
#Resuspended one colony.<br />
#Incuvated 16 hrs.<br />
</p><br />
<br />
----<br />
We decided to start another ligation plan. First we digestion Ag43(K346007) and dT(B0015) with EcoRI&SpeI and EcoRI&XbaI then ligate. Ligation product cut with XbaI&SpeI and HindiIII. HindiIII can cut the site which is exist in pSB1AK3 then we can confirm Ag43-dT(about 3000bp) and pSB1K3(3000bp: cutting fragment will be about 1000bp). Next we digest pT7-RBS on pSB1C3 with SpeI only and ligate X-Ag43-dT-S and S-pSB1C3-pT7-RBS-S. If that go well, we can get pT7-RBS-Ag43-dT on pSB1C3 plasmid DNA which has never been digested with PstI. <br />
<br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(K346007)<br />
</p><br />
</div><br />
<br />
==July 19th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and Ag43(K346007) which is inserted in pSB1C3.<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</P><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for Ag43-dT on pSB1T3[about 5400bp] and Ag43(K346007)[about 5200bp] plasmid extraction products.<br />
<br />
[[image:HokkaidoU2012 120719 Ag43-dT on pSB1T3 and Ag43(K346007) mini-prep product.jpg|thumb|plasmid extraction result]]<br />
<br />
From this plasmid extraction result, Ag43-dT on pSB1T3 showed obviously higher DNA band than we estimated. It means '''Ag43-dT and pSB1T3 were misligated.''' And Ag43(K346007), we thought '''Ag43(K346007) DNA band existed in correct area''' because this DNA has about 5200bp and plasmid DNA migrates more far than linear DNA. Additionally, one weak 10kbp band existed in Ag43(K346007) lane.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
We conducted two digestion.<br />
*Digestion for Ag43(K346007) cut with EcoRI and SpeI. This DNA is for confirmation of PstI restriction enzyme cutting site.<br />
*Digestion for Ag43(K346007) and dT(B0015) cut with EcoRI & SpeI and EcoRI & XbaI.<br />
But we cut dT PCR product which didn't have plasmid vector so we retry digestion of dT after.<br />
<br />
<br><br><br />
Insert Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|16 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Vector dT(E&X)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT(EcoRI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
dT(XbaI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7.8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120719 Ag43 cut with E&amp;S(each enzyme 0.1ul) and Ag43 cut with E&amp;S(each enzyme 1ul).jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed Ag43(K346007) was successfully digested into two fragments and low concentration of restriction enzyme cause unperfectly cut of plasmid extraction product. But there are correct two bands in Ag43 cutting result so restriction enzyme worked. And about dT digestion, XbaI worked in halfway. We thought this is because we didn't added BSA buffer into dT digestion mix.<br />
<br />
<br />
</p><br />
<br />
And we retried digestion of dT which was plasmid extraction and gel extracted products. Recipe was same as above(E&X).<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 20th==<br />
<div><br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion result done yesterday(dT cut with EcoRI and XbaI).<br />
Added 5 ul of EtBr and Pre-migrated for 30 min.<br />
Additionaly, to confirm the concentration of Ag43(K346007) gel extract result.<br />
<br />
[[image:HokkaidoU2012 120720 dTd- dTd+ Ag43(cut with E&amp;S)gelextract.jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed dT was successfully cut with EcoRI & SpeI.<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extraction products above.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 5 ul of DW.<br />
</P><br />
<br />
==Ligation==<br />
<p><br />
We ligated Ag43(purified at 7/17 and 7/20) as an insert and dT as vector.<br />
<br />
<br><br><br />
Ag43(7/20) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|2 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(7/17) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|4 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 21st==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion and ligation products yesterday.<br />
<br />
<br />
[[image:HokkaidoU2012 120721 ag43-d+ dt-d+ ligation print.jpg|thumb|Digestion and Ligation results]]<br />
<br />
There are three bands in ligation products(Ag43(7/20) + dT(on pSB1AK3))). Lower band would be digestion result which couldn't be ligated with dT. Middle band would be successfully ligaed DNA which have about 6k bp(Ag43 has 3.1k bp and dT on pSB1AK3 has 3.2k bp). And higher band would make something dimer, we thought. <br />
<br />
</p><br />
<br />
==Single colony isolation==<br />
<p><br />
Single colony isolation for incubated colonies spread yesterday. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extracted Ag43(E&S) gel extraction product for digestion of PstI Star activity confirmation.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis to confirm the concentration of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) Ethanol precipitation for PstItest.jpg|thumb|Ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion to confirm there are some PstI cutting site in Ag43(K346007)(cut with EcoRI and SpeI to remove PstI this parts potentially has).<br />
<br />
{|<br />
|Used DNA <br />
|K346007 cut with EcoRI and SpeI<br />
|-<br />
|Concentration(ng/ul)<br />
|25<br />
|-<br />
|Used DNA volume(ul) <br />
|2<br />
|-<br />
|theoretical ez value(ul)<br />
|0.02<br />
|}<br />
<br />
<br />
PstI = 0.1ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.9 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.2 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.2 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.8 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.5 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 1.0 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) cut with PstI(0.1, 0.2, 0.5, 1.jpg|thumb|Digestion result]]<br />
<br />
From this digestion, we confirmed there is at least one PstI cutting site in Ag43 fragment because these digestion results showed same bp and same number of cutting band.<br />
<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm how ligated Ag43 fragment(s) with dT on pSB1AK3.<br />
We used Ag43 last 700bp area as primer which had designed as sequencing primer. '''If three (two forward and one reverse )Ag43 were inserted, this primer can anneal to forward Ag43 as forward primer and reverse Ag43 as reverse primer then amplify about 766bp.''' If there are no reverse Ag43, DNA can't be amplified. <br />
<br><br><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
<br />
We failed amplification. <br />
Retry!<br />
<br />
==PCR==<br />
<p><br />
We did PCR written above once again. Change some point of reaction.<br />
We amplified Ag43(K346007) original DNA also as a control.<br><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
</p><br />
<br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 22nd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Results of digestion at 21st.<br /><br />
We expected to appear the band of about 700bp.<br />
So, we use 1% and 2% agarose gel.<br />
<br />
[[image:HokkaidoU_2012_120722_ag43_pcr-1%_print-1.jpg|thumb|PCR result image]]<br />
[[image:HokkaidoU2012_120722_ag43_pcr-2%_print.jpg|thumb|PCR result image]]<br />
<br />
<br /><br />
We can't find some band in 700~800bp...<br />
</p><br />
<br />
==Liquid Culture==<br />
<p><br />
Liquid culture for single colony isolated Ag43-dT on pSB1AT3.<br />
<br />
#Picked up one colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBA.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
Digested Ag43(K346oo7) plasmid extraction product with EcoRI and SpeI.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
</div></div><br />
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<h2>Notebook</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Notebook/Spring_Boot_Camp" class="hokkaidou-notebook-index" id="hokkaidou-notebook-bootcamp"><br />
<h5>Boot Camp</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Notebook/Lab_Diary" class="hokkaidou-notebook-index" id="hokkaidou-notebook-diary"><br />
<h5>Lab Diary</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Notebook/overall_protocols" class="hokkaidou-notebook-index" id="hokkaidou-notebook-protocols"><br />
<h5>Protocols</h5><br />
<p>Bible of our lab.</p><br />
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Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_3
Team:HokkaidoU Japan/Notebook/aggregation Week 3
2012-09-26T19:22:27Z
<p>Slecat: /* Digestion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 16th==<br />
<div><br />
Ag43, dT<br />
===Electrophoresis===<br />
<p><br />
Results of digestion at 15th.<br />
<br />
[[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&amp;P-1.jpg|thumb|Digestion result image]]<br />
Product:Ag43(K346007)=3120bp and 2070bp<br />pT7-RBS on pSB1K3=2247bp<br />
<br /><br />
We confirmed there are some kinds of restriction enzyme site in K346007 (digested with SpeI, PstI), and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.<br />
</P><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. Used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution.<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digested and extracted products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW.<br />
</p><br />
<br />
<br><br />
--------<br />
<br><br />
<br />
dT(B0015) would be amplified incorrectly and we couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.<br />
<br />
===PCR===<br />
<p><br />
PCR for dT(B0015)<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp_up forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(200bp_down Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]<br />
<br />
We migrated B0015 of plasmid extraction product, digestion product, and PCR product.<br />
dT done PCR would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
'''Digestion for dT which amplified with PCR.'''<br />
Digested with XbaI and PstI.<br />
<br /><br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
----<br />
<br />
Ag43<br />
<br />
'''Digestion result of Ag43 was incorrect'''. We digested Ag43 once more time.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for Ag43 with SpeI and PstI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43 DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120716 Ag43d- Ag43d+.jpg|thumb|Digestion result image]]<br />
<br />
There are same results with digestion result of recent. '''We thought PstI would cut different site'''.<br />
What is this 500bp fragment????<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50ul of DNA solution.<br />
</p><br />
<br />
----<br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.<br />
<br />
'''Colonies which have Ag43-dT on pSB1T3 were closely existed so we would picked up two or more colonies.''' <br />
#Picked up one (or tow?) colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBC and LBT.<br />
#Incuvated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 17th==<br />
<div><br />
<p><br />
Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3<br />
</p><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products incuvated from yesterday(16th).<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And plasmid extraction products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated.<br />
<br />
<br />
If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp.<br />
And if ligation and transformation, plasmid extraction were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp. <br />
<br />
[[image:HokkaidoU2012 120717 Ag43-Ag43d+(E&amp;S)-(Ag43-dT on pSB1T3)-(pT7-RBS on pSB1C3).jpg|thumb|Digestion and plasmid extraction result image]]<br />
<br />
From this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. '''would PstI not work correctly?''' <br />
<br />
<br />
'''We planed to use dT as vector and Ag43 as Insert.''' A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.<br />
<br />
<br />
About plasmid extraction products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought '''which means we failed single colony isolation then resuspended two another E.coli colonies''' another ligated DNA were transformed. <br />
In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.<br />
<br />
<br />
To confirm plasmid extraction products were really ligated correct DNA fragments, first we extracted Ag43-dT on pSB1T3(low bp band) from TBE gel and digested with EcoRI and PstI.<br />
</p><br />
<br />
----<br />
'''dT Vector plasmid change'''<br />
To use dT as a vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.<br />
<br />
==Digestion==<br />
<p><br />
Digestion to change the plasmid backbone.<br />
Used DNA solution as PCR product(did at 16th) and digestioned pSB1T3 were already exist.<br><br />
<br><br />
<br />
Digestion mix<br><br><br />
<br />
<br />
double digestion(EcoRI and PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717-dt-digestion-print.jpg|thumb|Digestion results]]<br />
<br />
We couldn't confirm digestion results.<br />
<br />
<br />
</p><br />
<br />
----<br />
pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3<br />
<br />
==Electrophoresis and gel extraction==<br />
<p><br />
[[image:HokkaidoU2012 120717-ag43-dt-psb1t3.jpg|thumb|plasmid extraction result]]<br />
'''Gel extraction for Ag43-dT on pSB1T3.'''<br><br />
We cut low bp band (see image below).<br />
Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not.<br />
<br><br><br />
<br />
'''Ag43-dT on pSB1T3(30 ng/ul)'''<br />
<br><br><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br><br />
'''pT7-RBS on pSB1C3(40 ng/ul)'''<br />
<br><br><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717 Ag43 dT(d-,d+E,d+P.jpg|thumb|Digestion result]]<br />
<br />
From this digestion results, <br />
*Cut with EcoRI,PstI and E&P(EcoRI and PstI) showed different base pair bands compared with d-(digestion minus).<br />
*Cut with E&P showed two DNA bands: about 2000bp and 500~1000 bp.<br />
*All of digestion products existed lower place than correct band.Correct DNA would show about 5000bp(EcoRI and PstI), 3000bp and 2000bp (E&P). <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 18th==<br />
<div><br />
<br />
<br />
'''We decided change plasmid backbone of dT pSB1AK3 to pSB1T3.'''<br><br />
If we cut Ag43-dT on pSB1AK3 with EcoRI and PstI to confirm insert DNA, DNA fragment base pair resemble each other(about 3000bp), so then we can't identify them.<br />
<br />
===digestion===<br />
<p><br />
<br />
'''Digestion to confirm what kind of restriction enzyme sites Ag43(K346007) has.'''<br />
<br><br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|- <br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120718-k346007-digestion print.jpg|thumb|Digestion result]]<br />
From this result, we doubted '''are there one or more another PstI resutriction enzyme site except BioBrick suffix'''?<br />
</p><br />
<br />
===PCR===<br />
<p><br />
PCR to confirm what DNA fragment is ligated to vector as insert.<br />
We used PCR for pT7-RBS on pSB1C3. So the result would be 80~90bp band.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Biobrick prefix forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(Biobrick suffix Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
[[image:HokkaidoU2012 120718 pT7-RBS on pSB1C3 EX-F PS-R PCR.jpg|thumb|PCR product]]<br />
</p><br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated(19hrs incubated) Ag43-dT on pSB1T3.<br />
<br />
#Prepared 2 ml LB.<br />
#Added Tet.<br />
#Resuspended one colony.<br />
#Incuvated 16 hrs.<br />
</p><br />
<br />
----<br />
We decided to start another ligation plan. First we digestion Ag43(K346007) and dT(B0015) with EcoRI&SpeI and EcoRI&XbaI then ligate. Ligation product cut with XbaI&SpeI and HindiIII. HindiIII can cut the site which is exist in pSB1AK3 then we can confirm Ag43-dT(about 3000bp) and pSB1K3(3000bp: cutting fragment will be about 1000bp). Next we digest pT7-RBS on pSB1C3 with SpeI only and ligate X-Ag43-dT-S and S-pSB1C3-pT7-RBS-S. If that go well, we can get pT7-RBS-Ag43-dT on pSB1C3 plasmid DNA which has never been digested with PstI. <br />
<br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(K346007)<br />
</p><br />
</div><br />
<br />
==July 19th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and Ag43(K346007) which is inserted in pSB1C3.<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</P><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for Ag43-dT on pSB1T3[about 5400bp] and Ag43(K346007)[about 5200bp] plasmid extraction products.<br />
<br />
[[image:HokkaidoU2012 120719 Ag43-dT on pSB1T3 and Ag43(K346007) mini-prep product.jpg|thumb|plasmid extraction result]]<br />
<br />
From this plasmid extraction result, Ag43-dT on pSB1T3 showed obviously higher DNA band than we estimated. It means '''Ag43-dT and pSB1T3 were misligated.''' And Ag43(K346007), we thought '''Ag43(K346007) DNA band existed in correct area''' because this DNA has about 5200bp and plasmid DNA migrates more far than linear DNA. Additionally, one weak 10kbp band existed in Ag43(K346007) lane.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
We conducted two digestion.<br />
*Digestion for Ag43(K346007) cut with EcoRI and SpeI. This DNA is for confirmation of PstI restriction enzyme cutting site.<br />
*Digestion for Ag43(K346007) and dT(B0015) cut with EcoRI & SpeI and EcoRI & XbaI.<br />
But we cut dT PCR product which didn't have plasmid vector so we retry digestion of dT after.<br />
<br />
<br><br><br />
Insert Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|16 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Vector dT(E&X)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT(EcoRI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
dT(XbaI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7.8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120719 Ag43 cut with E&amp;S(each enzyme 0.1ul) and Ag43 cut with E&amp;S(each enzyme 1ul).jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed Ag43(K346007) was successfully digested into two fragments and low concentration of restriction enzyme cause unperfectly cut of plasmid extraction product. But there are correct two bands in Ag43 cutting result so restriction enzyme worked. And about dT digestion, XbaI worked in halfway. We thought this is because we didn't added BSA buffer into dT digestion mix.<br />
<br />
<br />
</p><br />
<br />
And we retried digestion of dT which was plasmid extraction and gel extracted products. Recipe was same as above(E&X).<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 20th==<br />
<div><br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion result done yesterday(dT cut with EcoRI and XbaI).<br />
Added 5 ul of EtBr and Pre-migrated for 30 min.<br />
Additionaly, to confirm the concentration of Ag43(K346007) gel extract result.<br />
<br />
[[image:HokkaidoU2012 120720 dTd- dTd+ Ag43(cut with E&amp;S)gelextract.jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed dT was successfully cut with EcoRI & SpeI.<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extraction products above.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 5 ul of DW.<br />
</P><br />
<br />
==Ligation==<br />
<p><br />
We ligated Ag43(purified at 7/17 and 7/20) as an insert and dT as vector.<br />
<br />
<br><br><br />
Ag43(7/20) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|2 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(7/17) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|4 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 21st==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion and ligation products yesterday.<br />
<br />
<br />
[[image:HokkaidoU2012 120721 ag43-d+ dt-d+ ligation print.jpg|thumb|Digestion and Ligation results]]<br />
<br />
There are three bands in ligation products(Ag43(7/20) + dT(on pSB1AK3))). Lower band would be digestion result which couldn't be ligated with dT. Middle band would be successfully ligaed DNA which have about 6k bp(Ag43 has 3.1k bp and dT on pSB1AK3 has 3.2k bp). And higher band would make something dimer, we thought. <br />
<br />
</p><br />
<br />
==Single colony isolation==<br />
<p><br />
Single colony isolation for incubated colonies spread yesterday. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extracted Ag43(E&S) gel extraction product for digestion of PstI Star activity confirmation.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis to confirm the concentration of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) Ethanol precipitation for PstItest.jpg|thumb|Ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion to confirm there are some PstI cutting site in Ag43(K346007)(cut with EcoRI and SpeI to remove PstI this parts potentially has).<br />
<br />
{|<br />
|Used DNA <br />
|K346007 cut with EcoRI and SpeI<br />
|-<br />
|Concentration(ng/ul)<br />
|25<br />
|-<br />
|Used DNA volume(ul) <br />
|2<br />
|-<br />
|theoretical ez value(ul)<br />
|0.02<br />
|}<br />
<br />
<br />
PstI = 0.1ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.9 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.2 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.2 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.8 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.5 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 1.0 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) cut with PstI(0.1, 0.2, 0.5, 1.jpg|thumb|Digestion result]]<br />
<br />
From this digestion, we confirmed there is at least one PstI cutting site in Ag43 fragment because these digestion results showed same bp and same number of cutting band.<br />
<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm how ligated Ag43 fragment(s) with dT on pSB1AK3.<br />
We used Ag43 last 700bp area as primer which had designed as sequencing primer. '''If three (two forward and one reverse )Ag43 were inserted, this primer can anneal to forward Ag43 as forward primer and reverse Ag43 as reverse primer then amplify about 766bp.''' If there are no reverse Ag43, DNA can't be amplified. <br />
<br><br><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
<br />
We failed amplification. <br />
Retry!<br />
<br />
==PCR==<br />
<p><br />
We did PCR written above once again. Change some point of reaction.<br />
We amplified Ag43(K346007) original DNA also as a control.<br><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
</p><br />
<br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 22nd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Results of digestion at 21st.<br /><br />
We expected to appear the band of about 700bp.<br />
So, we use 1% and 2% agarose gel.<br />
<br />
[[image:HokkaidoU_2012_120722_ag43_pcr-1%_print-1.jpg|thumb|PCR result image]]<br />
[[image:HokkaidoU2012_120722_ag43_pcr-2%_print.jpg|thumb|PCR result image]]<br />
<br />
<br /><br />
We can't find some band in 700~800bp...<br />
</p><br />
<br />
==Liquid Culture==<br />
<p><br />
Liquid culture for single colony isolated Ag43-dT on pSB1AT3.<br />
<br />
#Picked up one colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBA.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
Digested Ag43(K346oo7) plasmid extraction product with EcoRI and SpeI.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_3
Team:HokkaidoU Japan/Notebook/aggregation Week 3
2012-09-26T19:20:11Z
<p>Slecat: /* Electrophoresis */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 16th==<br />
<div><br />
Ag43, dT<br />
===Electrophoresis===<br />
<p><br />
Results of digestion at 15th.<br />
<br />
[[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&amp;P-1.jpg|thumb|Digestion result image]]<br />
Product:Ag43(K346007)=3120bp and 2070bp<br />pT7-RBS on pSB1K3=2247bp<br />
<br /><br />
We confirmed there are some kinds of restriction enzyme site in K346007 (digested with SpeI, PstI), and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.<br />
</P><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. Used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution.<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digested and extracted products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW.<br />
</p><br />
<br />
<br><br />
--------<br />
<br><br />
<br />
dT(B0015) would be amplified incorrectly and we couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.<br />
<br />
===PCR===<br />
<p><br />
PCR for dT(B0015)<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp_up forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(200bp_down Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]<br />
<br />
We migrated B0015 of plasmid extraction product, digestion product, and PCR product.<br />
dT done PCR would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
'''Digestion for dT which amplified with PCR.'''<br />
Digested with XbaI and PstI.<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
----<br />
<br />
Ag43<br />
<br />
'''Digestion result of Ag43 was incorrect'''. We digested Ag43 once more time.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for Ag43 with SpeI and PstI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43 DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120716 Ag43d- Ag43d+.jpg|thumb|Digestion result image]]<br />
<br />
There are same results with digestion result of recent. '''We thought PstI would cut different site'''.<br />
What is this 500bp fragment????<br />
</p><br />
<br />
===Gel extraction===<br />
<p><br />
Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50ul of DNA solution.<br />
</p><br />
<br />
----<br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.<br />
<br />
'''Colonies which have Ag43-dT on pSB1T3 were closely existed so we would picked up two or more colonies.''' <br />
#Picked up one (or tow?) colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBC and LBT.<br />
#Incuvated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 17th==<br />
<div><br />
<p><br />
Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3<br />
</p><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products incuvated from yesterday(16th).<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And plasmid extraction products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated.<br />
<br />
<br />
If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp.<br />
And if ligation and transformation, plasmid extraction were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp. <br />
<br />
[[image:HokkaidoU2012 120717 Ag43-Ag43d+(E&amp;S)-(Ag43-dT on pSB1T3)-(pT7-RBS on pSB1C3).jpg|thumb|Digestion and plasmid extraction result image]]<br />
<br />
From this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. '''would PstI not work correctly?''' <br />
<br />
<br />
'''We planed to use dT as vector and Ag43 as Insert.''' A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.<br />
<br />
<br />
About plasmid extraction products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought '''which means we failed single colony isolation then resuspended two another E.coli colonies''' another ligated DNA were transformed. <br />
In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.<br />
<br />
<br />
To confirm plasmid extraction products were really ligated correct DNA fragments, first we extracted Ag43-dT on pSB1T3(low bp band) from TBE gel and digested with EcoRI and PstI.<br />
</p><br />
<br />
----<br />
'''dT Vector plasmid change'''<br />
To use dT as a vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.<br />
<br />
==Digestion==<br />
<p><br />
Digestion to change the plasmid backbone.<br />
Used DNA solution as PCR product(did at 16th) and digestioned pSB1T3 were already exist.<br><br />
<br><br />
<br />
Digestion mix<br><br><br />
<br />
<br />
double digestion(EcoRI and PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|dT PCR product<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717-dt-digestion-print.jpg|thumb|Digestion results]]<br />
<br />
We couldn't confirm digestion results.<br />
<br />
<br />
</p><br />
<br />
----<br />
pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3<br />
<br />
==Electrophoresis and gel extraction==<br />
<p><br />
[[image:HokkaidoU2012 120717-ag43-dt-psb1t3.jpg|thumb|plasmid extraction result]]<br />
'''Gel extraction for Ag43-dT on pSB1T3.'''<br><br />
We cut low bp band (see image below).<br />
Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).<br />
Got 50 ul of DNA solution.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not.<br />
<br><br><br />
<br />
'''Ag43-dT on pSB1T3(30 ng/ul)'''<br />
<br><br><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT DNA solution<br />
|3.3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13.7 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br><br />
'''pT7-RBS on pSB1C3(40 ng/ul)'''<br />
<br><br><br />
<br />
Control 1(EcoRI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
Control 2(PstI only)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|15.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
double digestion(EcoRI & PstI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS DNA solution<br />
|2.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|21 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120717 Ag43 dT(d-,d+E,d+P.jpg|thumb|Digestion result]]<br />
<br />
From this digestion results, <br />
*Cut with EcoRI,PstI and E&P(EcoRI and PstI) showed different base pair bands compared with d-(digestion minus).<br />
*Cut with E&P showed two DNA bands: about 2000bp and 500~1000 bp.<br />
*All of digestion products existed lower place than correct band.Correct DNA would show about 5000bp(EcoRI and PstI), 3000bp and 2000bp (E&P). <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 18th==<br />
<div><br />
<br />
<br />
'''We decided change plasmid backbone of dT pSB1AK3 to pSB1T3.'''<br><br />
If we cut Ag43-dT on pSB1AK3 with EcoRI and PstI to confirm insert DNA, DNA fragment base pair resemble each other(about 3000bp), so then we can't identify them.<br />
<br />
===digestion===<br />
<p><br />
<br />
'''Digestion to confirm what kind of restriction enzyme sites Ag43(K346007) has.'''<br />
<br><br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|- <br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120718-k346007-digestion print.jpg|thumb|Digestion result]]<br />
From this result, we doubted '''are there one or more another PstI resutriction enzyme site except BioBrick suffix'''?<br />
</p><br />
<br />
===PCR===<br />
<p><br />
PCR to confirm what DNA fragment is ligated to vector as insert.<br />
We used PCR for pT7-RBS on pSB1C3. So the result would be 80~90bp band.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Biobrick prefix forward primer)<br />
|1 ul<br />
|-<br />
|Reverse Primer(Biobrick suffix Reverse primer)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
[[image:HokkaidoU2012 120718 pT7-RBS on pSB1C3 EX-F PS-R PCR.jpg|thumb|PCR product]]<br />
</p><br />
<br />
===Liquid Culture===<br />
<p><br />
Liquid culture for single colony isolated(19hrs incubated) Ag43-dT on pSB1T3.<br />
<br />
#Prepared 2 ml LB.<br />
#Added Tet.<br />
#Resuspended one colony.<br />
#Incuvated 16 hrs.<br />
</p><br />
<br />
----<br />
We decided to start another ligation plan. First we digestion Ag43(K346007) and dT(B0015) with EcoRI&SpeI and EcoRI&XbaI then ligate. Ligation product cut with XbaI&SpeI and HindiIII. HindiIII can cut the site which is exist in pSB1AK3 then we can confirm Ag43-dT(about 3000bp) and pSB1K3(3000bp: cutting fragment will be about 1000bp). Next we digest pT7-RBS on pSB1C3 with SpeI only and ligate X-Ag43-dT-S and S-pSB1C3-pT7-RBS-S. If that go well, we can get pT7-RBS-Ag43-dT on pSB1C3 plasmid DNA which has never been digested with PstI. <br />
<br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(K346007)<br />
</p><br />
</div><br />
<br />
==July 19th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for Ag43-dT on pSB1T3 and Ag43(K346007) which is inserted in pSB1C3.<br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
</P><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for Ag43-dT on pSB1T3[about 5400bp] and Ag43(K346007)[about 5200bp] plasmid extraction products.<br />
<br />
[[image:HokkaidoU2012 120719 Ag43-dT on pSB1T3 and Ag43(K346007) mini-prep product.jpg|thumb|plasmid extraction result]]<br />
<br />
From this plasmid extraction result, Ag43-dT on pSB1T3 showed obviously higher DNA band than we estimated. It means '''Ag43-dT and pSB1T3 were misligated.''' And Ag43(K346007), we thought '''Ag43(K346007) DNA band existed in correct area''' because this DNA has about 5200bp and plasmid DNA migrates more far than linear DNA. Additionally, one weak 10kbp band existed in Ag43(K346007) lane.<br />
<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
We conducted two digestion.<br />
*Digestion for Ag43(K346007) cut with EcoRI and SpeI. This DNA is for confirmation of PstI restriction enzyme cutting site.<br />
*Digestion for Ag43(K346007) and dT(B0015) cut with EcoRI & SpeI and EcoRI & XbaI.<br />
But we cut dT PCR product which didn't have plasmid vector so we retry digestion of dT after.<br />
<br />
<br><br><br />
Insert Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|16 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Vector dT(E&X)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT(EcoRI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
dT(XbaI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(K346007)(E&S)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7.8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120719 Ag43 cut with E&amp;S(each enzyme 0.1ul) and Ag43 cut with E&amp;S(each enzyme 1ul).jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed Ag43(K346007) was successfully digested into two fragments and low concentration of restriction enzyme cause unperfectly cut of plasmid extraction product. But there are correct two bands in Ag43 cutting result so restriction enzyme worked. And about dT digestion, XbaI worked in halfway. We thought this is because we didn't added BSA buffer into dT digestion mix.<br />
<br />
<br />
</p><br />
<br />
And we retried digestion of dT which was plasmid extraction and gel extracted products. Recipe was same as above(E&X).<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 20th==<br />
<div><br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion result done yesterday(dT cut with EcoRI and XbaI).<br />
Added 5 ul of EtBr and Pre-migrated for 30 min.<br />
Additionaly, to confirm the concentration of Ag43(K346007) gel extract result.<br />
<br />
[[image:HokkaidoU2012 120720 dTd- dTd+ Ag43(cut with E&amp;S)gelextract.jpg|thumb|Digestion result]]<br />
<br />
From this result, we confirmed dT was successfully cut with EcoRI & SpeI.<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and<br />
got 50 ul of DNA solution.<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extraction products above.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 5 ul of DW.<br />
</P><br />
<br />
==Ligation==<br />
<p><br />
We ligated Ag43(purified at 7/17 and 7/20) as an insert and dT as vector.<br />
<br />
<br><br><br />
Ag43(7/20) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|2 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
Ag43(7/17) + dT<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Ag43<br />
|4 ul<br />
|-<br />
|dT<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 21st==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for digestion and ligation products yesterday.<br />
<br />
<br />
[[image:HokkaidoU2012 120721 ag43-d+ dt-d+ ligation print.jpg|thumb|Digestion and Ligation results]]<br />
<br />
There are three bands in ligation products(Ag43(7/20) + dT(on pSB1AK3))). Lower band would be digestion result which couldn't be ligated with dT. Middle band would be successfully ligaed DNA which have about 6k bp(Ag43 has 3.1k bp and dT on pSB1AK3 has 3.2k bp). And higher band would make something dimer, we thought. <br />
<br />
</p><br />
<br />
==Single colony isolation==<br />
<p><br />
Single colony isolation for incubated colonies spread yesterday. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for gel extracted Ag43(E&S) gel extraction product for digestion of PstI Star activity confirmation.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis to confirm the concentration of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) Ethanol precipitation for PstItest.jpg|thumb|Ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion to confirm there are some PstI cutting site in Ag43(K346007)(cut with EcoRI and SpeI to remove PstI this parts potentially has).<br />
<br />
{|<br />
|Used DNA <br />
|K346007 cut with EcoRI and SpeI<br />
|-<br />
|Concentration(ng/ul)<br />
|25<br />
|-<br />
|Used DNA volume(ul) <br />
|2<br />
|-<br />
|theoretical ez value(ul)<br />
|0.02<br />
|}<br />
<br />
<br />
PstI = 0.1ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.9 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.2 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.2 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.8 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 0.5 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI = 1.0 ul<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120721 Ag43(cut with E&amp;S) cut with PstI(0.1, 0.2, 0.5, 1.jpg|thumb|Digestion result]]<br />
<br />
From this digestion, we confirmed there is at least one PstI cutting site in Ag43 fragment because these digestion results showed same bp and same number of cutting band.<br />
<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm how ligated Ag43 fragment(s) with dT on pSB1AK3.<br />
We used Ag43 last 700bp area as primer which had designed as sequencing primer. '''If three (two forward and one reverse )Ag43 were inserted, this primer can anneal to forward Ag43 as forward primer and reverse Ag43 as reverse primer then amplify about 766bp.''' If there are no reverse Ag43, DNA can't be amplified. <br />
<br><br><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
<br />
We failed amplification. <br />
Retry!<br />
<br />
==PCR==<br />
<p><br />
We did PCR written above once again. Change some point of reaction.<br />
We amplified Ag43(K346007) original DNA also as a control.<br><br />
PCR recipe<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-forward primer<br />
|2 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
</p><br />
<br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 22nd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Results of digestion at 21st.<br /><br />
We expected to appear the band of about 700bp.<br />
So, we use 1% and 2% agarose gel.<br />
<br />
[[image:HokkaidoU_2012_120722_ag43_pcr-1%_print-1.jpg|thumb|PCR result image]]<br />
[[image:HokkaidoU2012_120722_ag43_pcr-2%_print.jpg|thumb|PCR result image]]<br />
<br />
<br /><br />
We can't find some band in 700~800bp...<br />
</p><br />
<br />
==Liquid Culture==<br />
<p><br />
Liquid culture for single colony isolated Ag43-dT on pSB1AT3.<br />
<br />
#Picked up one colony from single colony isolated plates by platinum loop.<br />
#Dipped into 2 ml of LBA.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
<br />
Digested Ag43(K346oo7) plasmid extraction product with EcoRI and SpeI.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:19:01Z
<p>Slecat: /* Electrophoresis */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed to incubate in liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br><br />
Digestion recipe<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br><br />
Ligation recipe<br><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br><br><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see July 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:17:28Z
<p>Slecat: /* Gel extraction */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed to incubate in liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br><br />
Digestion recipe<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br><br />
Ligation recipe<br><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br><br><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see July 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
<br />
'''Digestion result'''<br />
<br />
Digestion results for pT7-RBS-RFP-dT on pSB1C3<br />
(once digested with EcoRI and PstI)<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:16:07Z
<p>Slecat: /* Digestion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed to incubate in liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br><br />
Digestion recipe<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br><br />
Ligation recipe<br><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br><br><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
<br />
'''Digestion result'''<br />
<br />
Digestion results for pT7-RBS-RFP-dT on pSB1C3<br />
(once digested with EcoRI and PstI)<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:14:52Z
<p>Slecat: /* Plasmid extraction */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed to incubate in liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br><br />
Digestion recipe<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br><br />
Ligation recipe<br><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br><br><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
<br />
'''Digestion result'''<br />
<br />
Digestion results for pT7-RBS-RFP-dT on pSB1C3<br />
(once digested with EcoRI and PstI)<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:13:49Z
<p>Slecat: /* Electrophoresis results */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed to incubate in liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br><br />
Digestion recipe<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br><br />
Ligation recipe<br><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br><br><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
<br />
'''Digestion result'''<br />
<br />
Digestion results for pT7-RBS-RFP-dT on pSB1C3<br />
(once digested with EcoRI and PstI)<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Slecat
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_2
Team:HokkaidoU Japan/Notebook/aggregation Week 2
2012-09-26T19:13:14Z
<p>Slecat: /* Electrophoresis results */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==July 9th==<br />
<div><br />
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.<br />
===Colony PCR===<br />
<p><br />
Colony PCR for assembly products.<br />
#Picked up colony from LB plates by Autoclaved toothpicks.<br />
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PCR recipe<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|60<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
<br />
<br />
(Ag43 + dT)<br />
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|180<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 35<br />
</p><br />
<br />
===Electrophoresis results===<br />
<p><br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br /><br />
<br /><br />
<br />
pT7 + RBS on pSB1K3<br />
bbp-Insert-bbs:86bp<br />
<br />
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]<br />
Ag43 + dT on pSB1AK3<br />
bbp-Insert-bbs:3290bp<br />
<br />
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]<br />
<br />
<br />
We couldn't confirm insert DNA were really ligated with Vector or not.<br />
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. <br />
For plasmid extraction, we needed do liquid culture.<br />
</p><br />
<br />
===Incubate for plasmid extraction===<br />
<p><br />
#Prepared 1800 ul LB solutions.<br />
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).<br />
#Incubated for 15 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 10th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.<br />
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).<br />
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.<br />
</p><br />
<br />
<br />
pT7 + RBS on pSB1K3(Total 2247bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
<br />
Ag43 + dT on pSB1AK3(Total 6444bp)<br />
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]<br />
<br />
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.<br />
<br />
===Digestion===<br />
<p><br />
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br><br />
Digestion recipe<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pT7-RBS<br />
|1.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestion recipe<br><br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digestioned at 37c for 2 hrs.<br />
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]<br />
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]<br />
<br />
<br />
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion for 3A Assembly.<br />
<br />
<br><br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
<br />
pSB1C3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|} <br />
<br />
Reacted for 2 hrs at 37c.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 11th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
<br />
<br><br />
Ligation recipe<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|pSB1C3<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|8 ul<br />
|-<br />
|Total<br />
|16 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]<br />
<br />
===Transformation===<br />
<p><br />
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).<br />
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).<br />
#Incubated on ice for 30 min.<br />
#Heatshock for 1 min at 42c.<br />
#Added 200 ul of LB to transformed BL21(DE3) solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.<br />
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate. <br />
#Incubated for 23 hrs and 30 minutes.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 12th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for ligation product.Each products were reacted in recipes written below. <br />
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.<br />
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.<br />
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.<br />
#Ran PCR machine in recipe below.<br />
#Electrophoresis for confirmation of PCR results.<br />
<br />
<br />
PCR reaction solution<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|KapaTaq ready mix<br />
|5 ul<br />
|-<br />
|BioBrick prefix forward primer<br />
|0.5 ul<br />
|-<br />
|BioBrick suffix reverse primer<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''PCR recipe'''<br />
<br />
(pT7 + RBS)<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|94<br />
|30<br />
|-<br />
|3<br />
|68<br />
|90<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 40<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for some colonies used in colony PCR.<br />
#Prepared 200 ul LB solutions.<br />
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).<br />
#Incubated for 18 hrs and 30 min.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 13th==<br />
<div><br />
===Plasmid extraction===<br />
<p><br />
Plasmid extraction from colony No. 1~8.<br><br />
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.<br />
<br />
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm how DNA fragments ligated.<br />
<br />
<br><br />
Digestion Recipe<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA<br />
|4 ul<br />
|-<br />
|EcoRI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|} <br />
<br />
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 14th==<br />
<div><br />
===Ligation===<br />
<p><br />
Ligation for digestion fragments written above.<br />
<br><br />
Ligation recipe<br><br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert<br />
|5 ul<br />
|-<br />
|Vector<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.<br />
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation products written above.<br />
<br />
<br />
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB to transformed DH5&alpha; solution.<br />
#Pre-incubate for 2 hrs<br />
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.<br />
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.<br />
#Incubated for 21 hrs. <br />
<br />
<br />
<br><br />
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time.<br />
<br><br><br />
Showed the result.<br />
<br />
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Liquid culture===<br />
<p><br />
Liquid culture for Ag43(BBa_K346007)<br />
#Picked up one colony from plate done single colony isolation.<br />
#Dipped into LBC solution.<br />
#Incubated for 16 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 15th==<br />
<div><br />
===Gel extraction===<br />
<p><br />
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see 14th).<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6). <br />
<br><br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|BSA<br />
|1 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
EcoRI + PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
XbaI + SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
===Ethanol precipitation===<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.<br />
</p><br />
<br />
===Electrophoresis===<br />
<p><br />
Electrophoresis for digest-ethanol precipitation products.<br />
#Added 5 ul of EtBr.<br />
#Migrated for 30 min.<br />
<br />
<br />
'''Digestion result'''<br />
<br />
Digestion results for pT7-RBS-RFP-dT on pSB1C3<br />
(once digested with EcoRI and PstI)<br />
<br />
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]<br />
</p><br />
<br />
===Single colony isolation===<br />
<p><br />
Single colony isolation for transformation products of yesterday.<br />
#Picked up one colony from incubated LBC and LBT plate.<br />
#Spread it on LBC and LBT plate.<br />
#Incubated for 16 hrs.<br />
</p><br />
<br />
===Digestion===<br />
<p><br />
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).<br />
<br />
<br><br />
Ag43<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|9 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
dT<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1.1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
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