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- Annealing temperature of primers (Tm) should be around 60 C
- Check the secondary structure of the primers before you order them!
- no individual secondary structures i.e. hairpins
- no heterostructure with the forward and reverse primers together
- free energy of primers should be greater than -4 kCal
- GC content should be around 50% (40-60% is okay)
If using Phusion Master Mix, use this protocol: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531
Dilute your DNA to the following concentrations:
DNA Concentrations Template 0.1 - 1 ng/ul Forward Primer 10 uM Reverse Primer 10 uM - Set up a small box (e.g. empty pipette tip box) with ice and water. Your DNA and polymerase mix will go into this box before going into the the thermocycler in order to limit endonucelase activity.
Add the following DNA to a labeled 0.6ml PCR tube
DNA Volume Template 1 uL Forward Primer 500 nM Reverse Primer 500 nM Program the thermocycler as follows
Temperature Time 98 30s PAUSE 98 5s Tm 15 72 (15s)x(#kb) 72 5m 4 forever Wait for thermocycler to heat up
- Add 22.5uL of polymerase mix (Phusion Master Mix) to your DNA. Mix well and spin down. Transfer tubes to ice as soon as possible.
- Once the thermocycler has heated up to the right temperature (it should be paused at 98C), add tubes to thermocycler and resume PCR program.
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