Yeast DNA Prep-PDF

Protocol

1. grow up yeast culture to appropriate density (near saturation)
2. spin 1.5 ml 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:chloroform: isoamyl alcohol (25:24:1)
   • 200 ul (@200 mg) glass beads
5. 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.
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 the supernatant, and let dry upside down for 10 min.
10. 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)