grow up yeast culture to appropriate density (near saturation)
spin 1.5 mls of culture for 1 min in microfuge and aspirate off supernatant
resuspend pellet in 200 ul breaking buffer
wear gloves and add:
200 ul phenol:choloroform:isoamyl alcohol (25:24:1)
200 ul (@200 mg) glass beads
close cap tightly and vortex for 2.5 min.
Be careful when vortexing; label can be dissolved by the phenol.
Hold cap tightly so it doesn''t open or spill.
add 200 ul TE buffer and spin for 5 min, in microfuge
transfer 350 ml aqueous (top) layer to fresh eppendorf.
add 1 ml 95% ethanol and mix well, let sit for 10 minutes
spin for 2 min, take off supernatant, and let dry upside down 10 min.
resuspend pellet in 50 ul TE buffer or water.
You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli.
breaking buffer
2% (v/v) Triton X-100
1% (w/v) SDS
100 mM NaCl
10 mM Tris-Cl, pH 8.0
1 mM EDTA, pH 8.0
T.E. buffer (pH 8.0)
10 mM Tris-Cl, pH 8.0
1 mM EDTA, pH 8.0
chilled phenol:choloroform:isoamyl alcohol (25:24:1)
chilled 95% ethanol
acid-washed glass beads (Sigma, G 3753, See CPMB, 13.12.1)
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