Yeast DNA Prep protocol

From OpenWetWare

Solutions/reagents:

• yeast culture grown up to appropriate density
  
  (near saturation)
• 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)
• ice-cold phenol : chloroform : isoamyl alcohol
  
  (25:24:1)
• TE buffer
  
  (10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0)
• ice-cold 95% ethanol
• water
• glass beads

Equipment:

• Centrifuge
• Eppendorf tubes

Steps:

1. Measure out 1.5 ml of yeast culture grown up to appropriate density into Eppendorf tube (1).
   Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
   
2. Measure out 200 µl of breaking buffer into Eppendorf tube (1).
   Resuspend pellet by vortexing/by shaking vigorously.
   
3. Add 200 µl of ice-cold phenol : chloroform : isoamyl alcohol.
   Add 200 mg of glass beads.
   Wear gloves for this step!
   
4. Vortex the mixture for 2.5 mins .
   Be careful when vortexing; label can be dissolved by the phenol.
   Hold cap tightly so it doesn't open or spill.
   
5. Add 200 µl of TE buffer.
   Centrifuge at maximum speed for 5 mins at room temperature and aspirate out 350 µl of top layer.
   Transfer top aqueous layer into Eppendorf tube (2).
   Discard bottom layer.
   
6. Measure out 1 ml of ice-cold 95% ethanol into Eppendorf tube (2).
   Vortex the mixture for a few secs.
   Store at room temperature for 10 mins.
   
7. Centrifuge at maximum speed for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
   Stand the tube containing pellet for 10 mins in an inverted position on a paper towel to allow all of the fluid to drain away.
   

8. Option 1: Add 50 µl of TE buffer.
   (or)
   Option 2: Add 50 µl of water.
   

   Resuspend pellet by vortexing/by shaking vigorously.
   You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli.
   

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 30 mins