Protocol

1. grow up yeast culture to appropriate density (near saturation)

2. spin 1.5 mls of culture for 1 min in microfuge and aspirate off supernatant

3. resuspend pellet in 200 ul breaking buffer

4. wear gloves and add:

• 200 ul phenol:choloroform:isoamyl alcohol (25:24:1)

• 200 ul (@200 mg) glass beads

5. 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.

6. add 200 ul TE buffer and spin for 5 min, in microfuge

7. transfer 350 ml aqueous (top) layer to fresh eppendorf.

8. add 1 ml 95% ethanol and mix well, let sit for 10 minutes

9. spin for 2 min, take off supernatant, and let dry upside down 10 min.

10. 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.

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:choloroform:isoamyl alcohol (25:24:1)

• chilled 95% ethanol

• acid-washed glass beads (Sigma, G 3753, See CPMB, 13.12.1)