Thursday 11^th August

Lab work

Visualization

Transformation of DH5a cells with pPS16_009

By Léa

Dh5a cells were transformed with pPS16_009, or psB1c3 (control), or not transformed (control)using the usual protocol.

pPS16_010 extraction

By Alice

After screening colony screening PCR, 3 clones (clones 3, 7 and 8) transformed with pPS16_010 seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel.

Band size expected:

Migration of FRB plasmid

High Fidelity Phusion PCR of transformed cell with ligation 2 and 3

By Laetitia

PCR Phusion was performed on productct of ligation containing ligation 2 and 3 (gblocks) from 09/08/16. It was done following the usual protocol. For ligation 2 the primers used were IPS 123 and IPS 84. For ligation 3 the primers used were IPS 128 and IPS 83.

DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC 19

By Léa and Laetitia

PCR DreamTaq was performed on 6 clones of each transformed celles from 10/08/16. Each clone was plated and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel.

Phusion PCR on PSB1C3

By Alice

PSB1C3 was amplified with Phusion DNA polymerase following this protocol. Two primers (iPS41 and iPS42) were chosen. Annealing temperature was 70.9°C. Elongation step lasted 1min. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.

PCR products expected:

Migration of PSB1C3 amplification