Monday 11^th July

Lab work

Biobrick characterization

Electrocompetents and transformation by electroporation of BL21

By Charlène

BL21 cells were grown up to OD_600nm=7.14. They were then made competent and transformed with K1372001 extracted from clone 2 and pcl_TAA, pcl_TAG or pcl_Tq to create the following strains:

• BL21|K1372001+pcl_TAA
• BL21|K1372001+pcl_TAG
• BL21|K1372001+pcl_Tq

Bringing DNA closer

DH5α transformation with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN-

By Laetitia and Mathilde

DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- plasmids were resuspended with 5μL of sterilized water. 5 tubes were prepared :

• one control tube with only 50μL of DH5α heat-shock competent cells
• four tubes with 50μL of DH5α heat-shock competent cells + 1μL of plasmid

The regular heat-shock competent cells transformation protocol was used.

Transformed cells were plated in duplicata (10μL and 50μL) on 8 Petri dishes (spectinomycin 50μg/mL). Control cells were plated on another plate (spectinomycin 50μg/mL).

Visualization

Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007

By Alice

The colony screening PCR of the 08/07/16 is performed again. After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream the insertion site are chosen. PCR products expected below :

Electrophoresis of high fidelity PCR products

By Caroline

The PCR products from 08/07/2016 on clones pPS16_001, pPS16_002, pPS16_003, pPS16_005,pPS16_006 and pPS16_007 were migrated into a 0.8% agarose gel containing BET for 30min.

We obtained the same bands without offtarget but we can not send them to sequence because we do not have enough solution and concentration.

High fidelity PCR

By Caroline

The same PCR as 08/07/2016 was carry out but this time adapted to get 50µL final volume. Clones from transformations with two other plasmids were tested as well: pPS16_008 and pPS16_009.