Monday 4^th July

Lab work

Visualization

Ligation of Gblock 2-2

By Caroline and Lea

The size of the Gblock is 808pb so the ratio were up to 11.69 (>7). Gblock was softly centrifugate and took back with 75µL of TE. Then we vortexted the solution and let it at 50°C for 20 min. 1µL of ligase buffer, 1µL of ligase and 1µL of digested plasmids puc19 were put together with 7µL of Gblock2.2.

2 control solutions were made. The first with 1µL of digested plasmids puc19 and 9µL of water (Control 1), the second with 1µL of plasmids, 1µL of ligase and 1µL of water (Control 2).

Transformation of Gblocks in DH5α

By Caroline and Lea

6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005, pPS16_006. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted the 30/06/16. TODOajouter le lien The transformation protocol were followed increasing plasmids quantities (5µL instead of 1µL). After transformation, cells are spread on petri dishes containing LB + Ampicillin (50µg/mL) + Xgal + IPTG.

Digestion of plasmids

By Mathilde and Alice

The following plasmids were digested again with EcoR1 and HindIII using this protocol:

• pPS16_001 (from the 6 clones that were selected on 29/06/16)
• pPS16_003 (from the 6 clones that were selected on 29/06/16)
• pPS16_005 (from the 6 clones that were selected on 29/06/16)
• pPS16_006 (from the 6 clones that were selected on 29/06/16)

The digestion products were mixed as follow to be migrated on an agarose gel.

The DNA concentration was still insufficient meaning the extraction step was not efficient.

Bringing DNA closer

Plasmids extraction

By Naiane and Laetitia

Plasmids from the following strains were extracted with the kit "Charge Switch-Pro Plasmid Miniprep":

• DS-NMcas
• DS-SPcasN-
• DS-ST1casN-
• DS-TDcasN-

Then plasmids were digested with AvrII

The mix was incubated at 37°C for 1 hour. An electrophoresis was done (0.8% of agarose).