Tuesday 26^th July

Lab work

Visualization

High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009

By Alice

PCR peformed on July 25 with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The 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. 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 :

Low Fidelity Dreamtaq PCR of DH5α|pPS16_002

By Laetitia

We made a DreamTaq PCR on DH5α|pPS16_002 transformed cultures containing the Gblock 1.2. The PCR was done on 6 clones of transformed cells: 4 clones of the Petri dish from 19/07 and 2 clones of the Petri dish from 22/07. The usual protocol was used with TM at 57°C. These 6 clones were re-plated on a Petri dish containing LB, Ampicilin, IPTG and X-Gal.

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