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|K1372001 and pcl_TAA, pclTAG or pclTq

By Charlène

3 clones from each transformation condition (K1372001 and pcl_TAA, pclTAG or pclTq) 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

Total volume migration of PCR products 1.2 and Lig 4

By Terrence

A second migration on agarose gel was performed with the total volume of the PCR products 1.2 and 4. The migration allowed us to excise the DNA fragments from the agarose gel below and carry on with the Step 1 of the NucleoSpin Gel and PCR Clean-up kit protocol.

The excised bands from Step 1 were incubated with the buffer NT1 at 4°C overnight.

Gel extraction fo 1.2 and Lig 4

Low Fidelity DreamTaqPCR of DH5a|pPS16_016, DH5a|pPS16_010, DH5a|pPS16_012 and DH5a|pPS16_013

By Mathilde

PCR was performed on clones 1 to 5 for pPS16_016, clones 1,2,3,5 and 6 for pPS16_010, clones 1,2,3 and 6 for pPS16_012, and for clones 1,4 and 6 for pPS16_013. Both white ans blue colonies were picked for each plasmids.

Thus, the PCR mix was done for 17 tubes following the usual protocol. But 1µL of dNTPs were used instead of 2,5µL, and colonies were put in the water before the addition of the rest of the mix for each sample. Primers 1151 and 1152 were used, the initial denaturation time was 5min, and Tm = 57°c.

Each clone was put in liquide culture (LB 3mL + Ampicillin 50µg/mL)

Each PCR product was placed on agarose gel to migrate. 10µL of violet ladder was used, and the wells were filled with 1µL of PCR preparations.

PCR products expected were :

No PCR product presented the expected size.

Digestion of pPS16_013 clones 2 and 3

By Mathilde

The two plasmids were digested with the XbaI and PstI enzymes with the following protocol :

• 2µL of plasmid
• 0,5µL of each restriction enzyme
• 1µL of Green Fast Digestion Buffer
• 6 µL of sterile water

The two preparations were put for incubation during 5 min at 37°c, and then directly put to migrate.

Result of the PCR