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).

High fidelity PCRs on DS-TDcasN- and pZA21 plasmids

By Alice

2 PCRs with Q5® High-Fidelity 2X Master Mix were performed on DS-TDcasN- and pZA21 plasmids following this protocol. Ptet_R and Ptet_F primers were used to amplify pZA21.Link-TDdcas_F and Ter_TDdcas_R were used to amplify TD dcas9 sequence. Primers mix 100µM were used in the first PCR, and primers mix 10µM were used in the second. In both PCR, annealing temperature was 70°C. After amplification, 1 µL of loading dye is added to 5µL of each PCR products. Then 5 µL of this mix is put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 30min at 100V.

PCR products expected were :

A 2,1-sized band is observed for the PCR performed on pZA21 with primers mix 100µM. A -sized band is observed for the PCR performed on DS-TDcasN- with primers mix 10µM.

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 (1151_pheoR and 1152_pheoF) upstream and downstream the insertion site were chosen. Annealing temperature was 53°C. After amplification, 5 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 below :

For clones 3,4,5 and 6 transformed with pPS16_004, we can observe a 1Kb-sized band. These clones seem to have the plasmid pPS16_004 expected. All the same, for clones 2,4 and 5 transformed with pPS16_007, we can observe a 1 Kb-sized band. These clones seem to have the plasmid pPS16_007 expected.plasmid pPS16_007. Finally these clones are selected to perform a PCR with Q5 high fidelity DNA polymerase and both primers (1151_pheoR and 1152_pheoF). GBlocks 2.2 and 4.1 amplification with Q5 high fidelity DNA polymerase will be sent for sequencing.

Electrophoresis of high fidelity PCR products

By Caroline

The PCR products from 08/07/16 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 08/07/16 was carry out but this time adapted to get 50µL final volume. Clones from transformations with two other plasmids (28/06/2016) were tested as well: pPS16_008 and pPS16_009.