Team:HokkaidoU Japan/Notebook/plastic Week 9

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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 2nd==
+
===August 27th===
-
<div>
+
<div class="hokkaidou-section">
-
==Digestion==
+
[[image:HokkaidoU2012 120827 phaC digestion sugama.jpg|thumb|digestion result]]
-
<p>
+
[[image:HokaidoU2012 120827 RBS digestion sugama.jpg|thumb|digestion result]]
-
Digestion to divide BBa_K342001(PhaC) with XbaI and PstI.<br />And BBa_B0034(RBS) with SpeI and PstI (with three samples).
+
====Digestion====
-
<br /><br />
+
BBa_K342001(PhaC) was digested by XbaI and PstI.<br />And BBa_B0034(RBS) was digested by SpeI and PstI (with three samples).
PhaC (BBa_K342001)
PhaC (BBa_K342001)
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
Line 31: Line 31:
   |20 ul
   |20 ul
   |}
   |}
-
<br />
+
 
 +
 
RBS (BBa_B0034)
RBS (BBa_B0034)
-
<br />
+
N0. 1
-
N0.1
+
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
Line 55: Line 55:
   |25 ul
   |25 ul
   |}
   |}
-
<br /><br />
+
 
-
N0.2
+
 
 +
N0. 2
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
Line 77: Line 78:
   |25 ul
   |25 ul
   |}
   |}
-
<br />
 
-
N0.3<br />
+
 
 +
N0. 3
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
Line 100: Line 101:
   |25 ul
   |25 ul
   |}
   |}
-
</p>
 
-
==Gel extraction==
+
====Electrophoresis====
-
<p>
+
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.
-
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
+
-
</p>
+
-
</div></div>
+
-
<div class="hokkaidou-notebook-daily">
+
-
==August 22th==
+
-
<div>
+
-
==Mini-prep==
+
-
<p>
+
-
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR in 20th. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.  
+
-
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|mini-prep result]]
+
====Gel extraction====
 +
The digestion product was extracted. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
-
 
-
 
-
One of Ag43-dT on pSB1AK3 culture did not get myddy. And another one is only a little muddy.
 
-
We tried mini-prep to the latter, we god the 20 ul of DNA solution.
 
-
And then, we did electrophoresis the mini-prep products and (pBAD-RBS and pBAD) gel extract products.
 
-
 
-
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]
 
-
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]
 
-
</p>
 
-
 
-
==PCR==
 
-
<p>
 
-
PCR of pT7-RBS on pSB1C3.<br />
 
-
 
-
We used 4 kinds of primer set.
 
-
<br />
 
-
1 : EX-F , PS-R primer<br />
 
-
2 : EX-F , 200b down primer<br />
 
-
3 : 100b up , PS-R primer<br />
 
-
4 : 100b up , 200b down primer<br />
 
-
The density of primer solutions is 10 uM.
 
-
 
-
{|class="hokkaidou-table-pcr-reagent"
 
-
|-
 
-
|DNA solution
 
-
|1 ul
 
-
|-
 
-
|KOD-Plus-NEO(Taq polymerase)
 
-
|1 ul
 
-
|-
 
-
|dNTP
 
-
|5 ul
 
-
|-
 
-
|MgSO4
 
-
|3 ul
 
-
|-
 
-
|KOD-Plus-NEO Buffer
 
-
|5 ul
 
-
|-
 
-
|Forward Primer
 
-
|1 ul
 
-
|-
 
-
|Reverse Primer
 
-
|1 ul
 
-
|-
 
-
|DW
 
-
|33 ul
 
-
|-
 
-
|Total
 
-
|50 ul
 
-
|}
 
-
 
-
 
-
{|class="hokkaidou-table-pcr-time"
 
-
|-
 
-
|Number
 
-
|Degree
 
-
|Second
 
-
|-
 
-
|1
 
-
|94
 
-
|120
 
-
|-
 
-
|2
 
-
|98
 
-
|10
 
-
|-
 
-
|3
 
-
|58
 
-
|30
 
-
|-
 
-
|4
 
-
|68
 
-
|30
 
-
|-
 
-
|5
 
-
|4
 
-
|HOLD
 
-
|}
 
-
Cycle:2~4 x 45
 
-
 
-
 
-
 
-
[[image:|thumb|PCR result]]
 
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 23th==
+
===August 28th===
-
<div>
+
<div class="hokkaidou-section">
-
==Ethanol precipitation==
+
====Digestion====
-
<p>
+
Digestion to divide PhaA and PhaB with XbaI and PstI.<br />I digested PhaC(BBa_K342001) with these restriction sites and also XhoI to divide pSB1C3 into pieces, on which PhaC is, that is because the length of pSB1C3 is nearly PhaC.<br />And we digested PhaC (BBa_K342001) and pSB1C3 by XbaI and SpeI.<br /><br />
-
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution cut with XbaI & SpeI.
+
PhaA
-
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
+
{|class="hokkaidou-table-digestion"
-
#Centrifuged in 15000 rpm, 10 min at 4C.
+
-
#Remove supernatant and added 220 ul of 70% ethanol.
+
-
#Centrifuged in 15000 rpm, 10 min at 4C.
+
-
#Remove supernatant and air drying in room temperature then added 10 ul of DW.  
+
-
 
+
-
</p>
+
-
==Digestion==
+
-
<p>
+
-
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by cut with HindIII.
+
-
{|class="hokkaidou-table-digestion"
+
   |-
   |-
-
   |DNA solution ( 257ng/ul)
+
   |DNA solution (125 ng/ul)
-
   |9 ul
+
   |6.6 ul
   |-
   |-
-
   |HindIII(15U/ul)
+
   |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
   |1 ul
   |1 ul
   |-
   |-
Line 232: Line 131:
   |-
   |-
   |DW
   |DW
-
   |8 ul
+
   |9.4 ul
   |-
   |-
   |Total
   |Total
Line 239: Line 138:
-
{|class="hokkaidou-table-digestion"
+
PhaB
-
|-
+
{|class="hokkaidou-table-digestion"
-
|Number
+
-
|Degree
+
-
|Minute
+
-
|-
+
-
|1
+
-
|37
+
-
|180
+
-
|-
+
-
|2
+
-
|70
+
-
|15
+
-
|-
+
-
|3
+
-
|4
+
-
|HOLD
+
-
|}
+
-
 
+
-
 
+
-
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]
+
-
 
+
-
 
+
-
In this result, we confirmed that the pSB1AK3 was successfully digested with HindIII, but it was not clear how many pSB1AK3 were remaining as non-digested products.
+
-
 
+
-
</p>
+
-
==Gel extraction==
+
-
<p>
+
-
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
+
-
</p>
+
-
</div></div>
+
-
 
+
-
 
+
-
<div class="hokkaidou-notebook-daily">
+
-
==August 24th==
+
-
<div>
+
-
==Digestion==
+
-
<p>
+
-
Digestion of pT7-RBS on pSB1C3 with SpeI, Ag43-dT on pSB1AK3 with EcoRI & XbaI and pBAD-RBS with EcoRI & PstI.
+
-
 
+
-
 
+
-
Ag43-dT on pSB1AK3
+
-
 
+
-
 
+
-
 
+
-
E&X
+
-
{|class="hokkaidou-table-digestion"
+
   |-
   |-
-
   |DNA solution ( 120ng/ul)
+
   |DNA solution (125 ng/ul)
-
   |7 ul
+
   |4 ul
   |-
   |-
-
   |EcoRI
+
   |XbaI
   |1 ul
   |1 ul
   |-
   |-
-
   |XbaI
+
   |PstI
-
   |1
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
Line 300: Line 154:
   |-
   |-
   |DW
   |DW
-
   |9 ul
+
   |12 ul
   |-
   |-
   |Total
   |Total
Line 307: Line 161:
-
 
+
PhaC(BBa_K342001)
-
E (control)
+
{|class="hokkaidou-table-digestion"
-
{|class="hokkaidou-table-digestion"
+
   |-
   |-
-
   |DNA solution ( 120ng/ul)
+
   |DNA solution (125 ng/ul)
-
  |7 ul
+
-
  |-
+
-
  |EcoRI
+
-
  |1 ul
+
-
  |-
+
-
  |10xM buffer
+
-
  |2 ul
+
-
  |-
+
-
  |DW
+
   |10 ul
   |10 ul
-
  |-
 
-
  |Total
 
-
  |20 ul
 
-
  |}
 
-
 
-
 
-
 
-
X (control)
 
-
{|class="hokkaidou-table-digestion"
 
-
  |-
 
-
  |DNA solution ( 120ng/ul)
 
-
  |7 ul
 
   |-
   |-
   |XbaI
   |XbaI
-
   |1
+
   |1 ul
   |-
   |-
-
   |10xM buffer
+
   |PstI
-
  |2 ul
+
-
  |-
+
-
  |DW
+
-
  |10 ul
+
-
  |-
+
-
  |Total
+
-
  |20 ul
+
-
  |}
+
-
 
+
-
 
+
-
 
+
-
pBAD-RBS(E & S)
+
-
{|class="hokkaidou-table-digestion"
+
-
  |-
+
-
  |DNA solution ( 100ng/ul)
+
-
  |12 ul
+
-
  |-
+
-
  |EcoRI
+
   |1 ul
   |1 ul
   |-
   |-
-
   |SpeI
+
   |XhoI
-
   |1
+
   |5.1 ul
   |-
   |-
-
   |10xH buffer
+
   |10xM buffer
   |2 ul
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |4 ul
+
   |0.9 ul
   |-
   |-
   |Total
   |Total
Line 372: Line 186:
   |}
   |}
 +
====Electrophoresis====
 +
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.
 +
====Gel extraction====
 +
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
 +
</div></div>
-
pT7-RBS on pSB1C3 (SpeI)
+
<div class="hokkaidou-notebook-daily">
-
{|class="hokkaidou-table-digestion"
+
===August 29th===
-
  |-
+
<div class="hokkaidou-section">
-
  |DNA solution ( 20ng/ul)
+
====PHB polymer ethanolysis====
-
  |4 ul
+
We did ethanolysisãof PHB polymer for 4 hrs with sample 2~8.
-
  |-
+
-
  |SpeI
+
-
  |1 ul
+
-
  |-
+
-
  |10xM buffer
+
-
  |2 ul
+
-
  |-
+
-
  |DW
+
-
  |13 ul
+
-
  |-
+
-
  |Total
+
-
  |20 ul
+
-
  |}
+
 +
====Preparation for GC/MS====
 +
We did the preparation for GC/MS with sample 2~8.
-
 
+
====PCR====
-
{|class="hokkaidou-table-digestion"
+
We multiplied pSB1C3 by PCR.
 +
Used two different DNA polymerase, KOD-Plus-Neo and KAPA Taq.
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|Solution
 +
|Volume (ul)
|-
|-
-
|Number
+
|DNA
-
|Degree
+
|1
-
|Minute
+
|-
|-
 +
|Suffix-EX
|1
|1
-
|37
 
-
|180
 
|-
|-
-
|2
+
|Prefix-PS
-
|60
+
|1
-
|15
+
|-
|-
 +
|MgSO4
|3
|3
-
|4
 
-
|HOLD
 
-
|}
 
-
</p>
 
-
 
-
 
-
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]
 
-
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.
 
-
 
-
 
-
==Gel extraction==
 
-
<p>
 
-
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
 
-
 
-
</p>
 
-
 
-
==Ethanol Precipitation==
 
-
<p>
 
-
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.
 
-
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
 
-
#Centrifuged in 15000 rpm, 10 min at 4C.
 
-
#Remove supernatant and added 220 ul of 70% ethanol.
 
-
#Centrifuged in 15000 rpm, 10 min at 4C.
 
-
#Remove supernatant and air drying in room temperature then added 5 ul of DW.
 
-
</p>
 
-
 
-
==Ligation==
 
-
<p>
 
-
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.
 
-
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.
 
-
 
-
 
-
{|class="hokkaidou-table-ligation"
 
|-
|-
-
|Vector DNA
+
|dNTP
-
|4 ul
+
|5
|-
|-
-
|Insert DNA
+
|10x KOD-Plus-Neo Buffer
-
|4 ul
+
|5
 +
|-
 +
|KOD-Plus-Neo
 +
|1
|-
|-
|DW
|DW
-
|2 ul
+
|33
-
|-
+
-
|Ligation Mighty Mix
+
-
|10 ul
+
|-
|-
|Total
|Total
-
|20 ul
+
|50
|}
|}
-
Ligation reaction time was in detail below.
+
{|class="hokkaidou-table-pcr-time"
-
 
+
-
{|class="hokkaidou-table-ligation"
+
|-
|-
 +
|Number
|Degree
|Degree
-
|Minute
+
|Second
|-
|-
-
|16
+
|1
-
|30
+
|94
 +
|120
|-
|-
-
|65
+
|2
 +
|98
|10
|10
 +
|-
 +
|3
 +
|74
 +
|2
|-
|-
|4
|4
-
|Hold
+
|98
 +
|10
 +
|-
 +
|5
 +
|72
 +
|120
 +
|-
 +
|6
 +
|98
 +
|10
 +
|-
 +
|7
 +
|70
 +
|120
 +
|-
 +
|8
 +
|98
 +
|10
 +
|-
 +
|9
 +
|68
 +
|120
 +
|-
 +
|10
 +
|68
 +
|420
 +
|-
 +
|11
 +
|4
 +
|HOLD
|}
|}
-
 
+
Cycle1 : 2~3 x 5
-
 
+
Cycle2 : 4~5 x 5
-
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]
+
Cycle3 : 6~7 x 5
-
</p>
+
Cycle4 : 8~9 x 30
-
 
+
-
==Transformation==
+
-
<p>
+
-
Transformation for ligation product. We used E.coli strain DH5&alpha;.
+
-
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
+
-
#Incubated on ice for 30 min.
+
-
#Added 350 ul of LB.
+
-
#Incubated the cells for 2 hours at 37C.
+
-
#Prepared and Labeled two plastic plates with LBC.
+
-
#Plated 300 ul of the culture onto first dish and spread.
+
-
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
+
-
#Incubated the plates at 37C for  hours.
+
-
</p>
+
-
 
+
-
</div></div>
+
-
 
+
-
 
+
-
<div class="hokkaidou-notebook-daily">
+
-
==August 25th==
+
-
<div>
+
-
 
+
-
==Colony PCR==
+
-
<p>
+
-
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. 
+
{|class="hokkaidou-table-pcr-reagent"
{|class="hokkaidou-table-pcr-reagent"
 +
|Solution
 +
|Volume (ul)
|-
|-
-
|DNA solution
+
|DNA
-
|4 ul
+
|1
|-
|-
-
|Kapa-Taq(Taq polymerase)
+
|Suffix-EX (10 uM)
-
|5 ul
+
|2
|-
|-
-
|Forward Primer(ag43-f4 primer)
+
|Prefix-PS (10 uM)
-
|0.5 ul
+
|2
|-
|-
-
|Reverse Primer(200bp down primer)
+
|KAPA Taq
-
|0.5 ul
+
|25
 +
|-
 +
|DW
 +
|20
|-
|-
|Total
|Total
-
|10 ul
+
|50
|}
|}
-
 
{|class="hokkaidou-table-pcr-time"
{|class="hokkaidou-table-pcr-time"
Line 541: Line 332:
|-
|-
|3
|3
-
|53.0
+
|63.7
|30
|30
|-
|-
|4
|4
|72
|72
-
|60
+
|180
|-
|-
|5
|5
-
|72
 
-
|60
 
-
|-
 
-
|6
 
|4
|4
|HOLD
|HOLD
Line 558: Line 345:
Cycle:2~4 x 35
Cycle:2~4 x 35
-
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.
+
</div></div>
-
Desired product is about 695bp.
+
-
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]
+
<div class="hokkaidou-notebook-daily">
 +
===August 30th===
 +
<div class="hokkaidou-section">
 +
====Ethanol precipitation====
 +
Diegested phaA by XbaI and SpeI and RBS by SpeI were condensed by Ethanol precipitation.
-
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 (see August 24th) then noticed that the concentration of each ethanol precipitation products were 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.
+
====Ligation====
-
</p>
+
PhaA and RBS, PhaB and RBS, PhaB and pSB1C3 were ligated each other.<br />And the DNA were transformed into bacteria.
-
==Digestion==
+
====Colony PCR====
-
<p>
+
The length of PhaB on pSB1C3 was confirmed by colony PCR.<br />The result showed PhaB and pSB1C3 didn't ligate correctly.  
-
Digestion of vector DNA: pT7-RBS on pSB1C3 with SpeI. Not to leave the plasmid DNA as plasmid DNA, we cut the DNA in overnight.  
+
-
pT7-RBS on pSB1C3 (SpeI)
+
====Liquid Culture====
-
{|class="hokkaidou-table-digestion"
+
Incubation of bacteria holds RBS (BBa_B0034) - PhaC (K342001) was started.
-
  |-
+
-
  |DNA solution ( 20ng/ul)
+
-
  |4 ul
+
-
  |-
+
-
  |SpeI
+
-
  |1 ul
+
-
  |-
+
-
  |10xM buffer
+
-
  |2 ul
+
-
  |-
+
-
  |DW
+
-
  |13 ul
+
-
  |-
+
-
  |Total
+
-
  |20 ul
+
-
  |}
+
-
 
-
{|class="hokkaidou-table-digestion"
 
-
|-
 
-
|Number
 
-
|Degree
 
-
|Minute
 
-
|-
 
-
|1
 
-
|37
 
-
|600 (10 hours)
 
-
|-
 
-
|2
 
-
|60
 
-
|15
 
-
|-
 
-
|3
 
-
|4
 
-
|HOLD
 
-
|}
 
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 26th==
+
===August 31st===
-
<div>
+
<div class="hokkaidou-section">
-
==Gel extraction==
+
====Colony PCR====
-
<p>
+
We confirmed the length of the three constructs that transformed at August 30th.<br />The result showed RBS and PhaA were ligated correctly.<br />So the incubation was started.
-
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
+
[[image:HokkaidoU 120831 RBS phaA coloP edit (2).jpg|thumb|center|450px]]
-
</p>
+
====Plasmid extraction====
 +
Plasmid of RBS-PhaC were extracted.<br />And then we got 50ul DNA solution.
-
==Ethanol Precipitation==
+
====Liquid culture====
-
<p>
+
Incubation of bacteria holds dT (BBa_B0015) was started.
-
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.
+
-
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
+
-
#Centrifuged in 14000 rpm, 30 min at 4C.
+
-
#Remove supernatant and added 220 ul of 70% ethanol.
+
-
#Centrifuged in 15000 rpm, 15 min at 4C.
+
-
#Remove supernatant and air drying in room temperature then added 5 ul of DW.  
+
 +
</div></div>
-
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]
+
<div class="hokkaidou-notebook-daily">
-
</p>
+
===September 1st===
 +
<div class="hokkaidou-section">
-
==Ligation==
+
====Plasmid extraction====
-
<p>
+
Plasmids of RBS-PhaA and dT (BBa_B0015) were extracted.<br />And then we got 50ul DNA solution.
-
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.
+
[[image:HokkaidoU 120901 dT RBS-PhaA RBS-PhaC mini-prepç£ç© edit.jpg|thumb|center|600px|center|Fig. Extracted plasmids of dT and RBS-PhaA on pSB1A2]]<br />
-
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.  
+
1: dT on pSB1AK3 (About 3.3kbp)<br />
 +
2 to 4: RBS-PhaA on pSB1A2 (About 3.2kbp)<br />
 +
5 to 7: RBS-PhaC on pSB1A2 (About 4.1kbp)<br />We thought sample 4 is not ideal plasmid and trashed it.
 +
====Digestion====
 +
RBS-PhaA was digested by XbaI and SpeI restriction site to ligate with RBS-PhaC digested by SpeI site.
 +
[[image:HokkaidoU 120901 RBS-PhaA RBS-PhaC digestion.jpg]]
 +
*1 and 2 is digested RBS-PhaA on pSB1A2.
 +
*Upper fragment is vector, pSB1A2.
 +
*Lower one is an objective fragment, RBS-PhaA (About 1.2kbp).
 +
*And PhaB and pSB1C3 were digested with XbaI and SpeI site.<br/>We decided to try ligation PhaB with pSB1C3 again.
-
{|class="hokkaidou-table-ligation"
+
====Electrophoresis====
-
|-
+
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.
-
|Vector DNA
+
</div></div>
-
|0.25 ul
+
-
|-
+
-
|Insert DNA
+
-
|3 ul
+
-
|-
+
-
|DW
+
-
|1.75 ul
+
-
|-
+
-
|Ligation Mighty Mix
+
-
|5 ul
+
-
|-
+
-
|Total
+
-
|20 ul
+
-
|}
+
-
 
+
-
 
+
-
Ligation reaction time was in detail below.
+
-
 
+
-
{|class="hokkaidou-table-ligation"
+
-
|-
+
-
|Degree
+
-
|Minute
+
-
|-
+
-
|16
+
-
|30
+
-
|-
+
-
|65
+
-
|10
+
-
|-
+
-
|4
+
-
|Hold
+
-
|}
+
 +
<div class="hokkaidou-notebook-daily">
-
</p>
+
===September 2nd===
 +
<div class="hokkaidou-section">
-
==Transformation==
+
====Ethanol precipitation====
-
<p>
+
The digested DNAs, RBS-PhaA (No. 1), RBS-PhaA (No. 2), RBS-PhaC on pSB1A2, PhaB and pSB1C3 were concentrated by Ethanol precipitation.
-
Transformation for ligation product. We used E.coli strain DH5&alpha;.
+
-
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
+
-
#Incubated on ice for 30 min.
+
-
#Added 350 ul of LB.
+
-
#Incubated the cells for 2 hours at 37C.
+
-
#Prepared and Labeled two plastic plates with LBC.
+
-
#Plated 300 ul of the culture onto first dish and spread.
+
-
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
+
-
#Incubated the plates at 37C for  hours.  
+
-
</p>
+
====Ligation====
 +
RBS-PhaA (No. 1 and No. 2) was ligated with RBS-PhaC on pSB1A2.<br/>
 +
And PhaB was taken in pSB1C3.
 +
====Transformation====
 +
These ligated DNAs transformed into E. coli (strain: DH5&alpha;).<br/>
 +
And then we spread fungus liquid added LB on LB plates include antibiotics.
</div></div>
</div></div>

Latest revision as of 04:02, 27 September 2012

Contents

August 27th

digestion result
digestion result

Digestion

BBa_K342001(PhaC) was digested by XbaI and PstI.
And BBa_B0034(RBS) was digested by SpeI and PstI (with three samples). PhaC (BBa_K342001)

DNA solution (100 ng/ul) 12.5 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 3.5 ul
Total 20 ul


RBS (BBa_B0034) N0. 1

DNA solution (20.3 ng/ul) 14.3 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2.5 ul
DW 0.2 ul
Total 25 ul


N0. 2

DNA solution (15.6 ng/ul) 18.6 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2.5 ul
DW 1.9 ul
Total 25 ul


N0. 3

DNA solution (16.9 ng/ul) 17.2 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2.5 ul
DW 3.3 ul
Total 25 ul

Electrophoresis

We confirmed success of digestion by electrophoresis.
The DNA was extracted from TBE gel and we got DNA solution.

Gel extraction

The digestion product was extracted. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

August 28th

Digestion

Digestion to divide PhaA and PhaB with XbaI and PstI.
I digested PhaC(BBa_K342001) with these restriction sites and also XhoI to divide pSB1C3 into pieces, on which PhaC is, that is because the length of pSB1C3 is nearly PhaC.
And we digested PhaC (BBa_K342001) and pSB1C3 by XbaI and SpeI.

PhaA

DNA solution (125 ng/ul) 6.6 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 9.4 ul
Total 20 ul


PhaB

DNA solution (125 ng/ul) 4 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 12 ul
Total 20 ul


PhaC(BBa_K342001)

DNA solution (125 ng/ul) 10 ul
XbaI 1 ul
PstI 1 ul
XhoI 5.1 ul
10xM buffer 2 ul
DW 0.9 ul
Total 20 ul

Electrophoresis

We confirmed success of digestion by electrophoresis.
The DNA was extracted from TBE gel and we got DNA solution.

Gel extraction

Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

August 29th

PHB polymer ethanolysis

We did ethanolysisãof PHB polymer for 4 hrs with sample 2~8.

Preparation for GC/MS

We did the preparation for GC/MS with sample 2~8.

PCR

We multiplied pSB1C3 by PCR. Used two different DNA polymerase, KOD-Plus-Neo and KAPA Taq.

Solution Volume (ul)
DNA 1
Suffix-EX 1
Prefix-PS 1
MgSO4 3
dNTP 5
10x KOD-Plus-Neo Buffer 5
KOD-Plus-Neo 1
DW 33
Total 50


Number Degree Second
1 94 120
2 98 10
3 74 2
4 98 10
5 72 120
6 98 10
7 70 120
8 98 10
9 68 120
10 68 420
11 4 HOLD

Cycle1 : 2~3 x 5 Cycle2 : 4~5 x 5 Cycle3 : 6~7 x 5 Cycle4 : 8~9 x 30


Solution Volume (ul)
DNA 1
Suffix-EX (10 uM) 2
Prefix-PS (10 uM) 2
KAPA Taq 25
DW 20
Total 50
Number Degree Second
1 95 120
2 95 30
3 63.7 30
4 72 180
5 4 HOLD

Cycle:2~4 x 35

August 30th

Ethanol precipitation

Diegested phaA by XbaI and SpeI and RBS by SpeI were condensed by Ethanol precipitation.

Ligation

PhaA and RBS, PhaB and RBS, PhaB and pSB1C3 were ligated each other.
And the DNA were transformed into bacteria.

Colony PCR

The length of PhaB on pSB1C3 was confirmed by colony PCR.
The result showed PhaB and pSB1C3 didn't ligate correctly.

Liquid Culture

Incubation of bacteria holds RBS (BBa_B0034) - PhaC (K342001) was started.

August 31st

Colony PCR

We confirmed the length of the three constructs that transformed at August 30th.
The result showed RBS and PhaA were ligated correctly.
So the incubation was started.

HokkaidoU 120831 RBS phaA coloP edit (2).jpg

Plasmid extraction

Plasmid of RBS-PhaC were extracted.
And then we got 50ul DNA solution.

Liquid culture

Incubation of bacteria holds dT (BBa_B0015) was started.

September 1st

Plasmid extraction

Plasmids of RBS-PhaA and dT (BBa_B0015) were extracted.
And then we got 50ul DNA solution.

File:HokkaidoU 120901 dT RBS-PhaA RBS-PhaC mini-prepç£ç© edit.jpg
Fig. Extracted plasmids of dT and RBS-PhaA on pSB1A2

1: dT on pSB1AK3 (About 3.3kbp)
2 to 4: RBS-PhaA on pSB1A2 (About 3.2kbp)
5 to 7: RBS-PhaC on pSB1A2 (About 4.1kbp)
We thought sample 4 is not ideal plasmid and trashed it.

Digestion

RBS-PhaA was digested by XbaI and SpeI restriction site to ligate with RBS-PhaC digested by SpeI site. HokkaidoU 120901 RBS-PhaA RBS-PhaC digestion.jpg

  • 1 and 2 is digested RBS-PhaA on pSB1A2.
  • Upper fragment is vector, pSB1A2.
  • Lower one is an objective fragment, RBS-PhaA (About 1.2kbp).
  • And PhaB and pSB1C3 were digested with XbaI and SpeI site.
    We decided to try ligation PhaB with pSB1C3 again.

Electrophoresis

We confirmed success of digestion by electrophoresis.
The DNA was extracted from TBE gel and we got DNA solution.

September 2nd

Ethanol precipitation

The digested DNAs, RBS-PhaA (No. 1), RBS-PhaA (No. 2), RBS-PhaC on pSB1A2, PhaB and pSB1C3 were concentrated by Ethanol precipitation.

Ligation

RBS-PhaA (No. 1 and No. 2) was ligated with RBS-PhaC on pSB1A2.
And PhaB was taken in pSB1C3.

Transformation

These ligated DNAs transformed into E. coli (strain: DH5α).
And then we spread fungus liquid added LB on LB plates include antibiotics.


Retrieved from "http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_9"