Reshma P. Shetty at 13:02, 10 September 2009

Reshma P. Shetty — Thu, 10 Sep 2009 13:02:49 GMT

←Older revision		Revision as of 13:02, 10 September 2009
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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]]

JulianEskin at 21:03, 21 September 2005

JulianEskin — Wed, 21 Sep 2005 21:03:21 GMT

New page

Back to [[Protocols|OpenWetWare protocols]]

Back to [[Silver: Protocols|Silver Lab protocols]]

Back to [[Silver Lab]]

== 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 ddH<sub>2</sub>O 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 ddH<sub>2</sub>O
** 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

Silver: Colony PCR - Revision history

Reshma P. Shetty at 13:02, 10 September 2009

JulianEskin at 21:03, 21 September 2005