Team:HokkaidoU Japan/Notebook/aggregation Week 10
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(3rd colony-pcr) |
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+ | <div class="hokkaidou-notebook-daily"> | ||
+ | ==September 3rd== | ||
+ | <div> | ||
+ | ==Colony PCR of eCFP-RBS-pSB1A2== | ||
+ | <p> | ||
+ | |||
+ | |||
+ | {|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(200bp down primer) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |10 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |95 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |95 | ||
+ | |30 | ||
+ | |- | ||
+ | |3 | ||
+ | |53.0 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |5 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |6 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 35 | ||
+ | |||
+ | We used N1 (DW only) and N2 (ptetR-RBS-eCFP-dT-pSB1A2) as controls. | ||
+ | Desired product is about 776bp. | ||
+ | |||
+ | [[image:|thumb|Colony PCR result]] | ||
+ | |||
+ | |||
+ | We confirmed that about 70% of ligated DNA formed our desired construct. We selected No.2 and 5 colony for incubation and store No.7 and 8 colony mixture at 4C. | ||
+ | </div></div> | ||
+ | |||
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Revision as of 22:09, 2 September 2012
September 3rd
Colony PCR of eCFP-RBS-pSB1A2
DNA solution | 4 ul |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(ag43-f4 primer) | 0.5 ul |
Reverse Primer(200bp down primer) | 0.5 ul |
Total | 10 ul |
Number | Degree | Second |
1 | 95 | 120 |
2 | 95 | 30 |
3 | 53.0 | 30 |
4 | 72 | 60 |
5 | 72 | 60 |
6 | 4 | HOLD |
Cycle:2~4 x 35
We used N1 (DW only) and N2 (ptetR-RBS-eCFP-dT-pSB1A2) as controls. Desired product is about 776bp.
[[image:|thumb|Colony PCR result]]
We confirmed that about 70% of ligated DNA formed our desired construct. We selected No.2 and 5 colony for incubation and store No.7 and 8 colony mixture at 4C.
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