Team:Exeter/lab book/proto/12

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