Yeast DNA Prep

This protocol is used for recovering plasmids from yeast cultures.

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 ml phenol:choloroform:isoamyl alcohol (25:24:1)
   • 200 ml (@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 ml 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 ml TE buffer.

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)

From http://stausta.web.wesleyan.edu/mbb294/Expt3.html