Yeast DNA Prep protocol

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Solutions/reagents:

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

Measure out 1.5 ml of <a href="#yeast culture grown up to appropriate density" >yeast culture grown up to appropriate density</a> into Eppendorf tube (1).
Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.

Measure out 200 µl of <a href="#breaking buffer" >breaking buffer</a> into Eppendorf tube (1).
Resuspend pellet by vortexing/by shaking vigorously.

Add 200 µl of <a href="#ice-cold phenol : chloroform : isoamyl alcohol" >ice-cold phenol : chloroform : isoamyl alcohol</a>.
Add 200 mg of glass beads.
Wear gloves for this step!

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.

Add 200 µl of <a href="#TE buffer" >TE buffer</a>.
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.

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.

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.

Option 1: Add 50 µl of <a href="#TE buffer" >TE buffer</a>.
(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

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