Silver: Colony PCR

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Current revision (08:02, 10 September 2009) (view source)
 
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*Check product by running 10 µl PCR sample + 2 µl 6x DNA dye on 1% agarose gel
*Check product by running 10 µl PCR sample + 2 µl 6x DNA dye on 1% agarose gel
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[[Category:Yeast]] [[Category:Protocol]]

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Zymolyase Solution:

  • Regular stock of zymolyase is 10 mg/ml diluted in water, mix by inverting, not all will go into solution (filter it) store aliquots at -20°C (minimize freeze-thawing if possible)
  • Make 2.5 mg/ml zymolyase solution in 0.1 M sodium phosphate buffer pH 7.5 immediately before PCR experiment

Lysis of Yeast:

  • Aliquot 15 µl 2.5 mg/ml zymo solution (in 0.1 M sodium phosphate buffer pH 7.5) into thin-walled PCR tubes
  • Scrape small amount of yeast colony and resuspend in PCR tube (one lysis reaction when dilute will yield ~15 PCR reactions, only 5 µl is used for a template in each PCR re action)
  • Incubate on bench (RT) for 20 min
  • Place in PCR block and heat to 37°C for 5 min and then 95°C for 5 min

PCR Step:

  • Dilute the lysis solution 1:5 by adding 60 ul ddH2O to each PCR tube
  • Place 5 ul of this dilution into a fresh PCR tube
  • Add PCR mix (usually 45 ul total per reaction):
    • 2.5 µl 5’ Primer (10 µM)
    • 2.5 µl 3’ Primer (10 µM)
    • 5 µl Thermopol Buffer
    • 1 µl dNTPs (10 uM)
    • 33.5 µl ddH2O
    • 0.5 µl AmpliTaq or Vent DNA Polymerase
  • Run Lyse Program (LysAn50), takes about 5 hours
  • Check product by running 10 µl PCR sample + 2 µl 6x DNA dye on 1% agarose gel
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