Team:Paris Saclay/Notebook/July/25
Contents
Monday 25th July
Lab work
Visualization
Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002
By Mathilde
A DreamTaq PCR was made with the transformed culture pPS16_002 re-plated the day before. The usual protocol was followeed with Tm at 57°c and 5min for the initial denaturation.
We divided up the PCR mix in 6 PCR tubes and added in each one a different clone from the transformed culture. The picked colonies were re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000).
Results : PCR products expected were :
| Plasmid | pPS16_002 |
|---|---|
| Bande Size pb | 960 |
====High fidelity PCR on bacteria transformed with pPS16_001, pPS16_005, pPS16_008 and pPS16_009==== By Alice Plasmids pPS16_001, pPS16_005 and pPS16_009 containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol.
Primers used :
| Plasmids | gBlocks | Primer Forward | Primer Reverse |
|---|---|---|---|
| pPS16_001 | 1.1 | iPS138 | iPS120 |
| pPS16_005 | 3.1 | iPS138 | iPS126 |
| pPS16_009 | GFP 1-9 | iPS138 | iPS139 |
Annealing temperature was 72°C, and initial step was prolonged to 7 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.
PCR products expected were :
| Plasmids | gBlocks | Band size (bp) |
|---|---|---|
| pPS16_001 | 1.1 | 960 |
| pPS16_005 | 3.1 | 960 |
| pPS16_009 | GFP 1-9 | 862 |
