Team:HokkaidoU Japan/Notebook/aggregation Week 5

From 2012.igem.org

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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 30th==
+
===July 30th===
-
<div>
+
<div class="hokkaidou-section">
-
==digestion==
+
====Digestion====
-
<p>
+
I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.
-
I tried to digest the mini-prep products again, and after Ethanol precipitation, we did electrophoresis.
+
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]
-
</p>
+
====Transformation====
-
==transformation==
+
I think that the E. coli which we used transformation is BL21(DE3)pLysS.
-
<p>
+
So, the E. coli could grow on LBC plate.<br />
-
I think that the E. coli which we use transformation is BL21(DE3)pLysS.
+
I'm going to do transformation into DH5&alpha;.
-
So, the E. coli could multiplication increase on LBC plate.<br />
+
-
I'm going to do transformation , use DH5α.
+
-
Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(DH5α).
+
 
-
#Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
+
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.
-
#Incubated on ice for 30min.
+
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
-
#Added 200ul of LB then incubated the cells for 2 hours at 37C.
+
#Incubated on ice for 30 min.
 +
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
#Prepared and Labeled two petri dishes with LBK.
#Prepared and Labeled two petri dishes with LBK.
-
#Plate 200ul of the transformation onto first dish and spread.
+
#Plate 200 ul of the transformation onto first dish and spread.
-
#Added 450ul of LB to 50ul of the transformation and plated 200ul of it onto second dish and spread.  
+
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish.  
-
#Incubated the plates at 37C for 14 hours.
+
#Incubated the plates at 37C for 14 hrs.
-
</p>
+
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
 +
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 31th==
+
===July 31st===
-
<div>
+
<div class="hokkaidou-section">
-
==liquid culture==
+
====Liquid culture====
-
<p>
+
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.
-
#Added 2ml of LBK into culture tubes.
+
#Added 2 ml of LBK into culture tubes.
#Resuspended colonies.
#Resuspended colonies.
-
#Incubated the tubes at 37C for  
+
#Incubated the tubes at 37C for 18 hrs.
-
</p>
+
-
</div><div>
+
<br style="line-height: 0; clear: both;" />
 +
</div></div>
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 1st==
+
===August 1st===
-
<div>
+
<div class="hokkaidou-section">
-
==mini-prep==
+
====Plasmid extraction====
-
<p>
+
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.
-
mini-prep of pT7-RBS-Ag43-dT which had been cultivated from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.
+
[[image:HokkaidoU2012 120801 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]
-
[[image:|thumb|mini-prep result]]
+
-
</p>
+
-
</div><div>
+
-
==Digestion==
+
====Digestion====
-
<p>
+
'''pT7-RBS(20 ng/ul) '''=No. 9
-
'''pT7-RBS(20ng/ul) '''=⑨
+
<br />
-
 
+
SpeI using 10xH  
-
SpeI 10xH  
+
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution
   |DNA solution
-
   |4.5ul
+
   |4.5 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |DW
   |DW
-
   |3.5ul
+
   |3.5 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
-
SpeI 10xM  
+
 
 +
SpeI using 10xM
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution
   |DNA solution
-
   |4.5ul
+
   |4.5 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |DW
   |DW
-
   |3.5ul
+
   |3.5 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
-
'''pT7-RBS(30ng/ul) '''=⑩
+
'''pT7-RBS(30 ng/ul) '''=No. 10
-
 
+
<br />
-
SpeI 10xH  
+
SpeI using 10xH  
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution
   |DNA solution
-
   |3ul
+
   |3 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |DW
   |DW
-
   |5ul
+
   |5 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
-
SpeI 10xM  
+
SpeI using 10xM
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution
   |DNA solution
-
   |3ul
+
   |3 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |DW
   |DW
-
   |5ul
+
   |5 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
-
[[image:|thumb|digestion result]]
+
[[image:HokkaidoU2012 120801 pt7-rbs-digS.jpg|thumb|digestion result]]
-
We couldn't cut them exactry so we cut once more time.
+
We couldn't digested them exactly, so we tried to digest once more time.<br />
-
'''pT7-RBS(20ng/ul) '''=⑨
+
'''pT7-RBS(20 ng/ul) '''=No. 9<br />
SpeI 10xH  
SpeI 10xH  
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution
   |DNA solution
-
   |4.5ul
+
   |4.5 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
-
   |2ul
+
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |12.5ul
+
   |12.5 ul
   |-
   |-
   |Total
   |Total
-
   |20ul
+
   |20 ul
   |}
   |}
-
+
 
-
</p>
+
====Liquid culture====
-
==Liquid culture==
+
-
<p>
+
Liquid culture for pBAD-RBS on pSB1K3.
Liquid culture for pBAD-RBS on pSB1K3.
-
#Added 2ml of LBK into culture tube.
+
#Added 2 ml of LBK into culture tube.
#Scraped the surface of glycerol stock of construct.
#Scraped the surface of glycerol stock of construct.
-
#Incubated the tube at 33C for OOhrs.
+
#Incubated the tube at 33C.
-
</p>
+
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===August 2nd===
 +
<div class="hokkaidou-section">
 +
====Preparing chemical competent cell====
 +
Preparing chemical competent cell of BL21, JM109 and DH5&alpha;.
 +
Chemical competent cell made in each E. coli strains.
 +
<br /><br />
 +
Our competent cell Protocol
 +
#Did single colony isolation on LB plate.
 +
#Incubated the plate for 15-19 hours at 37C.
 +
#Lifted colony of E. coli into 2 ml LB.
 +
#Incubated at 37C for 12-16 hrs at 180-200 rpm.
 +
#Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.
 +
#Incubated cells at 20C (over 24 hrs) at 140 rpm.
 +
#Selected culture by measuring OD 600.
 +
#Incubated the 300 ml flask for 10 min on ice.
 +
#Transfered the culture into two 50 ml Falcon tubes.
 +
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.
 +
#Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)
 +
#Collected them to one tube.
 +
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.
 +
#Suspended the pellet in ice-cold 3.2 ml of TB.
 +
#Instilled 0.24 ml of DMSO in precipitant.
 +
#Incubated the 50 ml Falcon tube for 10 min on ice.
 +
#Divided 50 ul of solutions in each 0.5 ml tubes.
 +
#Freezed the suspension by liquid nitrogen.
 +
#Stored at â80C.
 +
 +
====Electrophoresis====
 +
Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)
 +
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|Electrophoresis result]]
 +
There were low concentration band above thick band. This thick band was same as digestion minus band.
 +
 +
====Digestion====
 +
Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |4 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |13 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===August 3rd===
 +
<div class="hokkaidou-section">
 +
 +
====Electrophoresis====
 +
Electrophoresis for the result of digestion.
 +
[[image:HokkaidoU2012 120803 pt7-rbs-dspe1.jpg|thumb|Electrophoresis result]]
 +
 +
====Gel extraction====
 +
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
 +
 +
====Ethanol precipitation====
 +
Ethanol precipitation for digestion and gel extraction product.
 +
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.
 +
#Centrifuged at 15000 rpm, 10 min at 4C.
 +
#Removed supernatant and added 220ul of 70% ethanol.
 +
#Centrifuged at 15000 rpm, 5 min at 4C.
 +
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.
 +
 +
====Ligation====
 +
Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|pT7-RBS
 +
|2 ul
 +
|-
 +
|Ag43-dT
 +
|4 ul
 +
|-
 +
|Ligation Mighty Mix(TAKARA)
 +
|6 ul
 +
|-
 +
|Total
 +
|12 ul
 +
|}
 +
 +
 +
Ligation reaction time was written below.
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Degree
 +
|Minute
 +
|-
 +
|16
 +
|30
 +
|-
 +
|65
 +
|10
 +
|-
 +
|4
 +
|Hold
 +
|}
 +
 +
 +
====Transformation====
 +
Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.
 +
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
 +
#Incubated on ice for 30min.
 +
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
 +
#Prepared and Labeled two petri dishes with LBK.
 +
#Spread 300 ul of the transformation onto first dish.
 +
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish.
 +
#Incubated the plates at 37C for 15 hrs 45 min.
 +
 +
====PCR====
 +
[[image:HokkaidoU2012 120803 Ag43 PCR(Forward=ag43-f4 Reverse=PS-R).jpg|thumb|PCR result]]
 +
PCR to confirm Ag43-f4 primer.
 +
{|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
 +
|-
 +
|Ag43-f4 primer
 +
|1 ul
 +
|-
 +
|PS-R 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
 +
|60
 +
|-
 +
|5
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:2~4 x 45
 +
 +
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 style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
 +
===August 4th===
 +
<div class="hokkaidou-section">
 +
====Colony PCR====
 +
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. 
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|-
 +
|DNA solution
 +
|4 ul
 +
|-
 +
|Kapa-Taq(Taq polymerase)
 +
|5 ul
 +
|-
 +
|Forward Primer(Ag43-f4 primer)
 +
|0.5 ul
 +
|-
 +
|Reverse Primer(PS-R primer)
 +
|0.5 ul
 +
|-
 +
|Total
 +
|10 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|95
 +
|120
 +
|-
 +
|2
 +
|95
 +
|30
 +
|-
 +
|3
 +
|58
 +
|30
 +
|-
 +
|4
 +
|72
 +
|60
 +
|-
 +
|5
 +
|72
 +
|60
 +
|-
 +
|6
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:2~4 x 35
 +
 +
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.
 +
Desired product is about 500~600bp.
 +
 +
[[image:HokkaidoU2012 120804 pt7-rbs-ag43-dt coloP.jpg|thumb|Colony PCR result]]
 +
 +
We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band.
 +
Next step, we resuspended these three colonies and incubated.
 +
 +
====Liquid culture====
 +
Liquid culture of colonies passed the colony PCR test.
 +
#Added 2 ml of LBK into culture tubes.
 +
#Resuspended 3 colonies.
 +
#Incubated the tubes at 37C for 13 hrs.
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===August 5th===
 +
<div class="hokkaidou-section">
 +
====Plasmid extraction====
 +
Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
 +
[[image:HokkaidoU2012 120805 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction results]]
 +
 +
====Digestion====
 +
Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.<br />
 +
pT7-RBS on pSB1K3
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |8 ul
 +
  |-
 +
  |SpeI
 +
  |2 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |8 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
pT7-RBS (once digestioned with SpeI)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |4 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |1 ul
 +
  |-
 +
  |DW
 +
  |4 ul
 +
  |-
 +
  |Total
 +
  |10 ul
 +
  |}
 +
 +
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |4 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |13 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
[[image:HokkaidoU2012 120805 pT7-RBS digestion SpeI (no.jpg|thumb|digestion result]]
 +
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?
 +
 +
====Gel extraction====
 +
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. 
 +
 +
====Sequencing====
 +
Sequencing to confirm what kind of DNA we made.
 +
 +
{|
 +
|DNA
 +
|primer
 +
|-
 +
|Ag43 plasmid extraction product
 +
|100bp-up forward primer,
 +
|ag43-f1,
 +
|ag43-f2,
 +
|ag43-f3,
 +
|ag43-f4
 +
|-
 +
|K542009
 +
|100bp-up forward primer,
 +
|ag43-f1,
 +
|ag43-f2,
 +
|ag43-f3,
 +
|ag43-f4
 +
|-
 +
|pT7-RBS on pSB1K3
 +
|100bp-up forward primer
 +
|-
 +
|pT7-RBS on pSB1C3
 +
|100bp-up forward primer
 +
|-
 +
|Ag43-dT on pSB1AK3
 +
|100bp-up forward primer,
 +
|ag43-f1,
 +
|ag43-f2,
 +
|ag43-f3,
 +
|ag43-f4
 +
|-
 +
|Ag43-dT on pSB1T3
 +
|100bp-up forward primer,
 +
|ag43-f1,
 +
|ag43-f2,
 +
|ag43-f3,
 +
|ag43-f4
 +
|-
 +
|pT7-RBS on pSB1K3
 +
|100bp-up forward primer
 +
|}
 +
 +
 +
Sequencing PCR
 +
{|class="hokkaidou-table-sequencing-pcr-reagent"
 +
|-
 +
|template DNA
 +
|1 ul
 +
|-
 +
|Ready Reaction Premix
 +
|1 ul
 +
|-
 +
|5x Sequencing Buffer
 +
|1.5 ul
 +
|-
 +
|H2O
 +
|5 ul
 +
|-
 +
|Primer(1 pmol/ul)
 +
|1.5 ul
 +
|-
 +
|Total
 +
|10 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|96
 +
|10
 +
|-
 +
|2
 +
|50
 +
|5
 +
|-
 +
|3
 +
|60
 +
|240
 +
|-
 +
|4
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:1~3 x 25
 +
 +
 +
To extract the PCR product, we did ethanol precipitation.
 +
<br />
 +
Ethanol precipitation for sequencing.
 +
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
 +
#Centrifuged at 15000 rpm, 15 min at 26C.
 +
#Removed supernatant and added 100ul of 70% ethanol.
 +
#Centrifuged at 15000 rpm, 5 min at 26C.
 +
#Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di.
 +
 +
Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)<br />
 +
We could not get the sequencing data.
 +
 +
====Digestion====
 +
Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |10 ul
 +
  |-
 +
  |HindIII
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |7 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
[[image:HokkaidoU2012 120805 Ag43-dT on pSB1AK3(d+ with XbaI &amp; SpeI) digestion with HindIII.jpg|thumb|digestion result]]
 +
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.
 +
 +
====Gel extraction====
 +
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
</div>
</div>
<br style="line-height: 0; clear: both;" />
<br style="line-height: 0; clear: both;" />
{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Latest revision as of 03:32, 27 September 2012

Contents

July 30th

Digestion

I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.

digestion result

Transformation

I think that the E. coli which we used transformation is BL21(DE3)pLysS. So, the E. coli could grow on LBC plate.
I'm going to do transformation into DH5α.


Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5α.

  1. Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Plate 200 ul of the transformation onto first dish and spread.
  6. Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish.
  7. Incubated the plates at 37C for 14 hrs.


July 31st

Liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.

  1. Added 2 ml of LBK into culture tubes.
  2. Resuspended colonies.
  3. Incubated the tubes at 37C for 18 hrs.


August 1st

Plasmid extraction

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.

Plasmid extraction result

Digestion

pT7-RBS(20 ng/ul) =No. 9
SpeI using 10xH

DNA solution 4.5 ul
SpeI 1 ul
10xH buffer 1 ul
DW 3.5 ul
Total 10 ul


SpeI using 10xM

DNA solution 4.5 ul
SpeI 1 ul
10xM buffer 1 ul
DW 3.5 ul
Total 10 ul


pT7-RBS(30 ng/ul) =No. 10
SpeI using 10xH

DNA solution 3 ul
SpeI 1 ul
10xH buffer 1 ul
DW 5 ul
Total 10 ul

SpeI using 10xM

DNA solution 3 ul
SpeI 1 ul
10xM buffer 1 ul
DW 5 ul
Total 10 ul
digestion result

We couldn't digested them exactly, so we tried to digest once more time.

pT7-RBS(20 ng/ul) =No. 9
SpeI 10xH

DNA solution 4.5 ul
SpeI 1 ul
10xM buffer 2 ul
DW 12.5 ul
Total 20 ul

Liquid culture

Liquid culture for pBAD-RBS on pSB1K3.

  1. Added 2 ml of LBK into culture tube.
  2. Scraped the surface of glycerol stock of construct.
  3. Incubated the tube at 33C.


August 2nd

Preparing chemical competent cell

Preparing chemical competent cell of BL21, JM109 and DH5α. Chemical competent cell made in each E. coli strains.

Our competent cell Protocol

  1. Did single colony isolation on LB plate.
  2. Incubated the plate for 15-19 hours at 37C.
  3. Lifted colony of E. coli into 2 ml LB.
  4. Incubated at 37C for 12-16 hrs at 180-200 rpm.
  5. Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.
  6. Incubated cells at 20C (over 24 hrs) at 140 rpm.
  7. Selected culture by measuring OD 600.
  8. Incubated the 300 ml flask for 10 min on ice.
  9. Transfered the culture into two 50 ml Falcon tubes.
  10. Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.
  11. Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)
  12. Collected them to one tube.
  13. Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.
  14. Suspended the pellet in ice-cold 3.2 ml of TB.
  15. Instilled 0.24 ml of DMSO in precipitant.
  16. Incubated the 50 ml Falcon tube for 10 min on ice.
  17. Divided 50 ul of solutions in each 0.5 ml tubes.
  18. Freezed the suspension by liquid nitrogen.
  19. Stored at â80C.

Electrophoresis

Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)

Electrophoresis result

There were low concentration band above thick band. This thick band was same as digestion minus band.

Digestion

Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.

DNA solution 4 ul
SpeI 1 ul
10xM buffer 2 ul
DW 13 ul
Total 20 ul


August 3rd

Electrophoresis

Electrophoresis for the result of digestion.

Electrophoresis result

Gel extraction

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

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 10 min at 4C.
  3. Removed supernatant and added 220ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 5 min at 4C.
  5. Removed supernatant and dried out at room temperature after that added 10 ul of DW.

Ligation

Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.

pT7-RBS 2 ul
Ag43-dT 4 ul
Ligation Mighty Mix(TAKARA) 6 ul
Total 12 ul


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold


Transformation

Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5α.

  1. Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30min.
  3. Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Spread 300 ul of the transformation onto first dish.
  6. Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish.
  7. Incubated the plates at 37C for 15 hrs 45 min.

PCR

PCR result

PCR to confirm Ag43-f4 primer.

DNA solution 1 ul
KOD-Plus-NEO(Taq polymerase) 1 ul
dNTP 5 ul
MgSO4 3 ul
KOD-Plus-NEO Buffer 5 ul
Ag43-f4 primer 1 ul
PS-R primer 1 ul
DW 33 ul
Total 50 ul


Number Degree Second
1 94 120
2 98 10
3 58 30
4 68 60
5 4 HOLD

Cycle:2~4 x 45

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.


August 4th

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(Ag43-f4 primer) 0.5 ul
Reverse Primer(PS-R primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 58 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls. Desired product is about 500~600bp.

Colony PCR result

We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band. Next step, we resuspended these three colonies and incubated.

Liquid culture

Liquid culture of colonies passed the colony PCR test.

  1. Added 2 ml of LBK into culture tubes.
  2. Resuspended 3 colonies.
  3. Incubated the tubes at 37C for 13 hrs.


August 5th

Plasmid extraction

Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction results

Digestion

Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.
pT7-RBS on pSB1K3

DNA solution 8 ul
SpeI 2 ul
10xM buffer 2 ul
DW 8 ul
Total 20 ul


pT7-RBS (once digestioned with SpeI)

DNA solution 4 ul
SpeI 1 ul
10xM buffer 1 ul
DW 4 ul
Total 10 ul


DNA solution 4 ul
SpeI 1 ul
10xM buffer 2 ul
DW 13 ul
Total 20 ul


digestion result

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?

Gel extraction

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.

Sequencing

Sequencing to confirm what kind of DNA we made.

DNA primer
Ag43 plasmid extraction product 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
K542009 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
pT7-RBS on pSB1K3 100bp-up forward primer
pT7-RBS on pSB1C3 100bp-up forward primer
Ag43-dT on pSB1AK3 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
Ag43-dT on pSB1T3 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
pT7-RBS on pSB1K3 100bp-up forward primer


Sequencing PCR

template DNA 1 ul
Ready Reaction Premix 1 ul
5x Sequencing Buffer 1.5 ul
H2O 5 ul
Primer(1 pmol/ul) 1.5 ul
Total 10 ul


Number Degree Second
1 96 10
2 50 5
3 60 240
4 4 HOLD

Cycle:1~3 x 25


To extract the PCR product, we did ethanol precipitation.
Ethanol precipitation for sequencing.

  1. Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 15 min at 26C.
  3. Removed supernatant and added 100ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 5 min at 26C.
  5. Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di.

Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)
We could not get the sequencing data.

Digestion

Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.

DNA solution 10 ul
HindIII 1 ul
10xM buffer 2 ul
DW 7 ul
Total 20 ul
digestion result

From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.

Gel extraction

Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.



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