Lidstrom:Colony PCR

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Revision as of 15:36, 2 February 2012 by Janet B. Matsen (talk | contribs) (General:)
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  • Use Taq. It is cheap. Do not use Phusion. It is ~$1/50uL rxn. Taq is less accurate & less fast but that's fine.
  • The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC.
  • The annealing temperature is up to you. For VF2 & VR 54-56oC works well.
  • The primer concentration varies in different people's recipes.
  • One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used.
  • Do rxns in 10-25 uL. Too small of a volume can result in overloading without noticing.
  • If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion.
  • Janet is using NEB's OneTaq quick-load (not hot start) & their recommended protocol
  • Stay tuned for an image of what "overloading" can look like!