Silver:ColonyPCR

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