Team:Exeter/lab book/proto/12

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Latest revision as of 23:50, 26 September 2012

Protocol 12

3-Step PCR

Make-up a 50µL total solution in a PCR tube of:

  • 5µL of 10ng/µL genomic DNA template
  • 1µL dNTPs
  • 1µL Forward Primer (@100pmol/µL)
  • 1µL Reverse Primer (@100pmol/µL)
  • 10µL High-Fidelity Phusion 5x Buffer
  • 1µL High-Fidelity Phusion polymerase
  • 31µL MilliQ H2O

Run a three-step PCR experiment using the following settings:/p>

  • Stage 1: 98oC for 5 minutes for 1 cycle
  • Stage 2: 98oC for 30 seconds followed by 65oC for 20 seconds, followed by 72oC for 30 seconds, all for 30 cycles
  • Stage 3: 72oC for 5 minutes for 1 cycle

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