Protocol
- grow up yeast culture to appropriate density (near saturation)
- spin 1.5 ml 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:chloroform: isoamyl alcohol (25:24:1)
- 200 ul (@200 mg) glass beads
- close the cap tightly and vortex for 2.5 min.
- Be careful when vortexing; the label can be dissolved by the phenol.
- Hold the 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 the supernatant, and let dry upside down for 10 min.
- resuspend the 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.
Materials
- 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:chloroform: isoamyl alcohol (25:24:1)
- chilled 95% ethanol
- acid-washed glass beads (Sigma, G 3753, See CPMB, 13.12.1)