Wednesday 13^th July

Lab work

Getting 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.

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

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)

For the primers used, Tm is 57°C

PCR Program:

Culture of DH5αlpPS16_008 on petri dish

By Laetitia