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.

Result of the extraction of pPS16003, pPS16004 and GFP1-9_PSB1C3 (clones 2 and 3) and result of the migration of gBlock 2-3-4

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 amplify them 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

Gel Extraction of 1.2 and Lig 4

By Terrence

The PCR products of gblock 1.2 and ligation 4 were purified on gel using the NucleoSpin Gel and PCR Clean-up from Macherey-Nagel.

Gel extraction fo 1.2 and Lig 4