IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/10/16

PCR QCM - 2nd part

• I digested the PCR product with DpnI for 1h, then I transformed either 1, 3 or 5µL of digested product in NEB and MG1655 cells.
• I add 200µL of LB and then let recovering at 37°C for 1h.

I plated everything on LB+Amp, and let it grow O/N at 37°C.

Soil sample analysis

I took 3 sample of soil from the sidewalk, at 3 different places, that weight about 20-30g. I add 25mL of distilled water and vortex hard for couples of minutes. I let settled over night. This morning I took in duplicate 50µL, 25µL, 15µL, 5µL of supernatant for sample. I add water up to 50µL. I put this at 99°C for 10min, then I spin it down for 5min, and I took 5µL of the supernatant. So, I ran the PCR with : • 5µL of supernatant • 15µL of water • 2,5µL of primers (VF2 & VR) • 25µL of PCR MM (contain taq, dNTP, ..)

PCR pgm :

• 95°C - 1'
• 95°C - 30"
• 50°C - 30"
• 72°C - 1'
• 72°C - 10'
• 10°C - inf

-> 30 cycles

• Pictures of the gels :

Soil sample test