Silver: Colony PCR

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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&deg;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&deg;C for 5 min and then 95&deg;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