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Transfer enough cells to make 10ul 20mM NaOH slightly cloudy. 100C for 10 min, use 0.5-1ul for PCR.

or could try this instead (from Biotechniques. 2011 May; 50(5): 325–328.) (worked well on first attempt)

  1. Pick one yeast colony from the plate or spin down 100-200 μl of liquid yeast culture (OD600=0.4). Suspend cells in 100 μl of 200mM LiOAc, 1 % SDS solution.
  2. Incubate for 5 minutes at 70°C.
  3. Add 300μl of 96-100 % ethanol, vortex.
  4. Spin down DNA and cell debris at 15 000 g for 3 minutes.
  5. (Can Skip this step, just get rid of as much EtOH as possible - flick open tube) - Wash pellet with 70 % ethanol
  6. Dissolve pellet in 100 μl of H2O or TE and spin down cell debris for 15 seconds at 15 000 g.
  7. Use 1 μl of supernatant for PCR.





For some reason the super easy below protocol hasn't been working anymore... 

This is the super quick and easy yeast colony PCR protocol. It has worked great for me numerous times with different primers.


MaterialsEdit

  • I've successfully performed this method using GemTaq polymerase
  • Other colony PCR protocols may mention the addition of betaine or DMSO as required, but neither was necessary when I tested it. However, different primers or template (particularly GC rich regions) might be aided by DMSO or betaine addition.
  • If you have some extra purified DNA (e.g., from a phenol chloroform extraction aka 'smash and grab' ), you may want to include that DNA as a control in case the PCR does not work, then you will know whether there is an issue with the PCR in general or specifically with the colony PCR .
  • Yeast on a plate (from liquid could probably work out too? maybe spin down the culture and toothpick some cells? update the page if you try from liquid culture and it works)

ProcedureEdit

Setup PCR reactions as normal (25ul total PCR rxns worked fine for me, probably 10ul would work) Edit

Pick some yeast from a plate and put into the PCR rxn tube (I made the PCR solution reasonably cloudy with yeast) Edit

Perform PCR as normal except include a 95C initial step for 15-30min (I should double check the time I did that worked, but I think it was 20min - use the 'BWcolony' pcr protocol on the machine).Edit

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