https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=Rapid_Isolation_of_Genomic_DNA_from_Yeast 2026-05-30T23:23:03Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=Rapid_Isolation_of_Genomic_DNA_from_Yeast&diff=610&oldid=prev 2012-02-21T16:32:30Z

<p>copied protocol from emily kauffman</p> <p><b>New page</b></p><div>[[Category:Genotyping]]<br /> [[Category:Yeast]]<br /> [[Category:PCR]]<br /> [[Category:DNA]]<br /> <br /> ==Materials==<br /> ===Yeast DNA Prep Buffer===<br /> * 2 ml 10% Triton X-100<br /> * 1 ml 10% SDS<br /> * 1 ml 1M NaCl<br /> * 100 µl 1M Tris pH8<br /> * 20 µl 0.5M EDTA<br /> * Fill to 10 ml with ddH2O<br /> <br /> ==Protocol==<br /> # Grow 10ml yeast cultures to saturation in YEPD 24C (for 2 overnights)<br /> # Collect the cells by centrifugation – 5min. 4,500 rpm<br /> # Resuspend the cell pellet in 0.5ml sterile ddH2O<br /> # Transfer to a 1.5ml microfuge tube and collect by centrifugation for 1 min, 10,000 rpm<br /> # Decant the supernatant and briefly vortex tube to resuspend pellet in residual liquid<br /> # Add 0.2 ml of Buffer A<br /> # Add 0.2 ml of phenol:chloroform:isoamyl alcolohol, and 0.3 grams of acid washed glass beads<br /> # Mix in the Tomy Mixer on highest speed for 4 minutes then add 0.2 ml TE pH 8<br /> # Centrifuge for 5 minutes, 10,000 rpm, transfer the aqueous layer to a fresh tube. Add 1 ml of 200 proof Ethanol. Mix by inversion gently.<br /> # Centrifuge for 2 minutes, 10,000 rpm. Discard the supernatant. Resuspend pellet in 0.4 ml of TE and 15 µl of 2 mg/ml RNaseA (DNase Free). Incubate 5 minutes at 37C. Add 8µl of 5M NH4 acetate plus 1 ml 200 proof Ethanol. Mix by inversion gently<br /> # Centrifuge for 2 minutes, discard the supernatant. Air dry the pellet and resuspend in 50µl TE</div>

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