Yeast DNA Prep

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From http://stausta.web.wesleyan.edu/mbb294/Expt3.html
From http://stausta.web.wesleyan.edu/mbb294/Expt3.html
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[[Category:Protocol]]
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[[Category:In vitro]]
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[[Category:Yeast]]
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[[Category:DNA]]

Revision as of 13:10, 9 May 2007

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 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)

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

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