Wednesday 13^th July

Lab work

Bringing DNA closer

Glycerol stocks for DH5α tra,sformed with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN-

By Laetitia

8 stocks were made: 2 clones (Cl 1 and Cl 2) by cas:

• Cl1 and Cl2 of DS-NMcas
• Cl1 and Cl2 of DS-SPcasN-
• Cl1 and Cl2 of DS-ST1casN-
• Cl1 and Cl2 of DS-TDcasN-

For 1 glycerol stock:

• 1mL of liquid culture
• 500 μL of glycerol 60%

Amplification of DS_SPcasN, DS_TDcasN, pZA21 and pZA31 by Phusion PCR

By Caroline

The different parts needed for de getting DNA closer tool were amplify by high fidelity PCR using Phusion following usual protocol with a TM at 60°C. Specific primers were used to amplify. These primers will allow the Gibson assembly afterward : Ptet_R and Ptet_F for the plasmids, Link-TDdcas_F and Ter_TDdcas_R for DS_TDcasN, Tet-SPdcas_F and Link-SPdcas_R for DS_SPcasN.

Biobrick characterization

BL21 electrocompetent cells preparation and transformation

By Mathilde and Charlène

We did an electro-transformation of BL21 with :

• pcl_TAA + K1372001 (time constant equal to 5.9ms)
• pcl_TAG + K1372001 (time constant equal to 6 ms)
• pcl_Tq + K1372001 (time constant equal to 6 ms)

Cells were displayed on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a dish with 50µL of cells and another with 500µL of cells.

Visualization

PCR of DH5αlpPS16_008 with Dream Taq and electrophoresis

By Laetitia

Preparation of the PCR mix (total of 200μL ):

• 20 μL of Dream Taq Green Buffer
• 20 μL of dNTP (10 mM)
• 8 μL of primer 1151 (10 mM)
• 8 μL of primer 1152 (10 mM)
• 1,04 μL of Dream Taq
• 142,96 μL of Nuclease free water

We divided up the PCR mix in 7 PCR tubes: put 25 μL of the mix in each tube.

For each tube (x7 different clones) : We picked 1 white clone in the petri dish containing DH5αlpPS16_008 (from 12/07/16) and soaked it in the tube (with PCR mix)

PCR was performed using this program :

We used a TM at 57°C. The initial denaturation lasted 5 min.

The products from the PCR were migrated on gel by electrophoresis during 20 min. We used the blue scale.

Culture of DH5αlpPS16_008 clones on petri dish

By Laetitia

We divided a Petri dish in 7(for 7 clones from DH5αlpPS16_008). Just after putting the DH5αlpPS16_008 clone in the PCR tube, spread the cells with the stick on the part corresponding to the clone on the petri dish.

We left it grow at room temperature.

High fidelity PCR on bacteria transformed with pPS16_007

By Alice

High fidelity PCR perfomed on the 12/07/16 did not give expected results. Thus high fidelity colony PCR is performed again on bacteria transformed with pPS16_007 following this protocol. 1151_pheoR and 1152_pheoF primers were used. Annealing temperature was 60°C and initial denaturation was prolonged to 10 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were 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 :

No PCR products were observed.

Colony screening PCR on bacteria transformed with pPS16_004

By Alice and Caroline

Clones of bacteria transformed with pPS16_004 selected on the 11/07/16 did not grow on petri dishes. That why we performed again colony screening PCR on these bacteria. After transformation, only white bacteria were selected (blue white screen). They were expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase was performed following this protocol. Clones selected for PCR were 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 and initial denaturation was prolonged to 6 min. 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 were:

No PCR products were observed, despite the experiment was performed twice.