Rapid DNA from yeast/PCR
vvsvetlov at utmem1.utmem.edu
Thu Feb 8 21:06:16 EST 1996
It's been a few times we had questions about isolation of DNA from yeast
for PCR. Following protocols were adapted from the white book - Methods in
Yeast Genetics by Kaiser, Michaelis and Mitchell, CSHL Press, 1994. Both
are sufficiently quick but unlike other "quick" and "1-step" protocols one
finds everywhere actually work.
RAPID RELEASE OF PLASMID DNA FROM YEAST
1. Pellet cells from 1.5 ml of o/night culture of yeast in Eppendorf
centrifuge. Discard the supernatant.
2. Add 0.2 ml of 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl
(8.0), 1mM Na-EDTA to the pellet. Add 0.2 ml of phenol: chloroform:
isoamyl alcohol (25:24:1) and 0.3 ml of acid washed glass beads.
3. Vortex at high speed for 2 min.
4. Centrifuge for 5 min.
5. Transfer top layer to a new tube - can be used directly in
transformation of E. coli (2-5 ul) or yeast (15-20 ul), concentrated or
precipitated with isopropanol.
ISOLATION OF GENOMIC DNA FROM YEAST
1. Pellet cells from 10 ml of o/night culture, resuspend the pellet in 0.5
ml of water, transfer the suspension into Eppendorf tube and pellet again.
Discard the supernatant.
2. Follow step 2 from the above protocol.
3. Vortex for 3-4 min at high speed. Add 0.2 ml of TE (8.0).
4. Centrifuge for 5 min. Transfer the top layer into a new tube; add 1 ml
of 100% (96%) ethanol. Mix by inversion.
5. Pellet DNA (5 min at top speed). Discard the supernatant, air-dry the
pellet. Resuspend the DNA in 0.4 ml of TE (8.0), add 10 ul of RNAse A (10
mg/ml). Incubate 30 min at 370C. Add 10 ul of 4 M ammonium acetate and 1
ml of 100% (96%) ethanol. Mix by inversion.
6. Pellet DNA as above. Discard the supernatant, air-dry the pellet and
resuspend DNBA in 50 ul of TE (8.0). Concentration of DNA 0.1-1 ug/ul.
Correction - my biblio info I emailed to couple of folks was with error -
the three guys named above are authors not editors of the book.
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