Monday 18^th July

Preparation of LB solid and liquid stock

By Caroline and Terrence

- 1L of LB liquid : 20g/L powder LB + 1L MilliQ water
- 500mL of LB solid : 500mL of LB liquid + 7.5g/L of Agar
The solutions were put in autoclave for sterilization.

Bringing DNA closer

Electrophoresis of PCR products DS-SPcasN-, DS-TDcasN-, pZA21 and pZA31

By Caroline

PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0.8% agarose gel.

Migration of SP,TD, pZA21 and pZA31

Biobrick characterization

LB culture of electro-transformed BL21 cells (cf. protocol)

By Mathilde

Each transformation: BL21|pcl_TAA + K1372001, BL21|pcl_TAG + K1372001 and BL21|pcl_Tq + K1372001 was added to 3mL of LB and one or two antibiotics.

Three conditions were tested for each transformation :

• 15µL of Chloramphenicol (30µg/mL)
• 5 µL of Streptomycin (50µg/mL)
• 15µL of Chloramphenicol (30µg/mL) + 5 µL of Streptomycin (50µg/mL)

Cultures were put in incubation at 37°C, 180 rpm.

Visualization

Electrophoresis of PCR products pPS16_004

By Caroline

PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0.8% agarose gel

Transformation of bacteria with pPS16_004 and pPS16_007

By Alice, Terrence and Mathilde

PCR made on bacteria transformed with pPS16_004 and pPS16_007 did not give expected results. We supposed that bacteria used for these PCRs did not still have the plasmid of interest. That is why we performed another transformation of DH5α competent cells with pPS16_004 and pPS16_007. We transformed cells following this protocol. We add 5μl of sterile H₂O to the ligation product rest and used the whole for the transformation. Finally, we spread the cells on Petri dishes (LB + Amp 50μg/mL + X-Gal 0.25μL/mL + IPTG 0.1 μL/mL) in duplicate .