User:Andrew Perry/Protocols/Basic PCR protocol

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See also:

http://openwetware.org/wiki/Cfrench:KodPCR

http://openwetware.org/wiki/Silver:_PCR

http://polbase.neb.com/ -- properties of many DNA polymerases

Recipe for 50 μL reaction

  • 10 µL polymerase buffer
  • 1 µL 10 mM dNTPs (or 4 ul of 2.5 mM) (keep on ice)
  • 0.5 µL Forward Primer (100 µM)
  • 0.5 µL Reverse Primer (100 µM)
  • 1 µL plasmid DNA template (typically between 1 pg and 10 ng total)
  • 0.5 µL DNA polymerase (Taq/Phusion/Pfu/Vent/KOD) (keep in cold block at -20 °C)
  • 32.5 µL sterile distilled water (up to 50 µL)

Extension temp for:

  • Taq, = 72 degrees (60 seconds/kb, 45-60 for < 1 kb)
  • Phusion = 72 degrees (15–30 seconds/kb)
  • Pfx, Pfu = 68 degrees
  • Vent = 74 degrees (for 1000 bp/min rate)
  • KOD polymerase = 72 degrees (~ 100 bp/ second = ~ 6000 bp/min)

Example cycle:

Start:

  • 98 °C for 30 sec.

Cycle (x 25):

  • 98 °C for 10 sec.
  • 55 °C for 15 sec. (use Tm, or 3 degrees above the primer Tm for annealing temperature)
  • 68 or 72 °C for 15-30 sec./kb (eg, 60 sec for 1 kb)

End:

  • 72 °C for 5-10 min.
  • 4 °C forever

(or to save power and prevent condensation final temperature can be changed to 10 degrees, or 4 degrees for 5 min then 22 degrees forever. The product should still be stable)

Colony PCR

  • Pick up a single colony using a sterile pipette tip and put it into 100 µL sterile water (scrape the tip against the side of the tube and pipette up and down a little to ensure the colony goes into the water). Give it a quick vortex & save these tubes at 4 °C.
  • Add 1 μL of this resuspended colony as the DNA template to 19 uL of PCR master mix above.
  • Use primers that will anneal to your target sequence.
  • Do 30x PCR cycles rather than 25x - 55 °C is a reasonable 'default' annealing temperature.
  • Run reactions on an agarose gel as usual and look for an amplified band at the size of your expected product.
  • Inoculate an overnight culture (containing appropriate antibiotic) with 20 - 50 µL of your resuspended colony solution & grow at 37 °C shaking overnight - this culture can be used to produce glycerol stocks and plasmid minipreps to be sequenced.
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