Team:Paris Saclay/protocols/pcr

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10 mM dNTPs
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dNTPs 10 mM
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1 μl
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3. Determine the number of oligo by electrophoresis.
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3. Verify correct amplification by agarose gel electrophoresis.

Latest revision as of 09:24, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

PCR for the genomic DNA of bacteria

1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.


Component Volume/50µL reaction Volume/20µL reaction

H2O

Add to 50 μl

Add to 20 μl

5x Phusion HF Buffer High-fidelity

10 μl

4 μl

dNTPs 10 mM

1 μl

0.4 μl

Primer A

x μl

x μl

Primer B

x μl

x μl

Template DNA: Genomic DNA

x μl

x μl

Phusion DNA polymerasse

0.5 μl

0.2 μl



2. Program the PCR machine according to the Table 2.


Cycle step Temperature Time Cycle

Initial denaturation

95 - 98 °C

30 s – 3 min

1

Denaturation

95 - 98 °C

10 – 30 s

25-30

Annealing

variable

30 s

25-30

Extension

72°C

30 s – 2 min

25-30

Final extension

72°C

10min

1

Final extension

4-8°C

hold

1

3. Verify correct amplification by agarose gel electrophoresis.



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