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 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

BioStream version

Following is the Yeast DNA Prep protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Yeast DNA Prep protocol

Source Code

Yeast DNA Prep protocol - source code