Monday 25^th July

Lab work

Visualization

Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002

By Mathilde and Caroline

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 :

The protocol was made again with 6 other white colonies from de same Petri dish.

High fidelity Q5 PCR on bacteria transformed with pPS16_004, pPS16_007 and pPS16_008

By Caroline

High fidelity Q5 PCR was carried out on colonies transformed with pPS16_004, pPS16_007 and pPS16_008 following the usual protocol adapted for 50µL final volume with puc19 universal primers. This was made in order to have more PCR products for pPS16_004 and pPS16_007 to send to sequencing. For pPS16_008 we had to send it again using PCR from new transformed colonies because we did not found the previous one.

High fidelity PCR on bacteria transformed with pPS16_001, pPS16_005 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. Specific primers were used for each plasmids (table below). Annealing temperature calculated 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 :