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

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(PCR QCM - 2nd part)
(a)
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I plated everything on LB+Amp, and let it grow O/N at 37°C.
I plated everything on LB+Amp, and let it grow O/N at 37°C.
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==Soil sample analysis==
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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.
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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.
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So, I ran the PCR with :
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• 5µL of supernatant
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• 15µL of water
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• 2,5µL of primers (VF2 & VR)
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• 25µL of PCR MM (contain taq, dNTP, ..)
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PCR pgm :
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95°C - 1'
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95°C - 30"
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50°C - 30"
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72°C - 1'
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72°C - 10'
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4°C -\infty
==Soil sample test==
==Soil sample test==

Revision as of 15:14, 16 October 2012

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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' 4°C -\infty

Soil sample test

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