Wednesday 17^th August

Lab work

Visualization

Extraction of pPS16003, pPS16004 and GFP1-9_PSB1C3 (clones 2 and 3)

By Charlène

Clones 2 and 3 of GFP1-9_PSB1C3, and pPS16003, pPS16004 were extracted with the Plasmid MiniPrep kit.

The extracts were put for migration on a 0.8%agarose gel with BET.

Purification of gBlocks 2, 3 and 4

By Terrence

The purification was carried out following the usual protocol.

Cloning with pJET the gBlock 1.2, FRB, FKBP and SgNM

By Léa and Naiane

The cloning was carried out using a new protocol which uses pJET as cloning vector.

A heat shock transformation was made on the cloning samples using the following protocol

Culture of BL21

By Charlène

3 clones from each transformation condition were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL). Then each solution was split into 3x150µL and 350µL of mix of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture. Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM.

Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation

By Alice

Q5 PCR was performed directly on gBlocks to try to amplified it following this protocol. Primers used were:

Annealing temperature was 70°C. Elongation step was up to 1 min. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.

PCR products expected were :

Migration of gBlocks and ligation