Team:Exeter/lab book/proto/7

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Latest revision as of 23:48, 26 September 2012

Protocol 7

Two-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 two-step PCR experiment using the following settings:

  • Stage 1: 98oC for 30 seconds for 1 cycle

  • Stage 2: 98oC for 10 seconds followed by 72oC for 20 seconds for 30 cycles

  • Stage 3: 72oC for 5 minutes for 1 cycle

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