Yeast DNA Prep protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="yeast culture grown up to appropriate density">yeast culture grown up to appropriate density <tab><div style="margin-right: 600px;">(near saturation)</div></a></li><li> <a name="breaking buffer">breaking buffer <tab><div style="margin-right: 600px;">(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)</div></a></li><li> <a name="ice-cold phenol : chloroform : isoamyl alcohol"> ice-cold phenol : chloroform : isoamyl alcohol <tab><div style="margin-right: 600px;">(25:24:1)</div></a></li><li> <a name="TE buffer">TE buffer <tab><div style="margin-right: 600px;">(10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0)</div></a></li><li>ice-cold 95% ethanol</li><li>water</li><li>glass beads</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><li>Measure out 1.5 ml of <a href="#yeast culture grown up to appropriate density" >yeast culture grown up to appropriate density</a> into an Eppendorf tube. Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 200 µl of <a href="#breaking buffer" >breaking buffer</a>. Resuspend the pellet by vortexing/by shaking vigorously. </li><li>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! </li><li>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. </li><li>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 an Eppendorf tube. Discard bottom layer. </li><li>Add 1 ml of ice-cold 95% ethanol. Vortex the mixture for a few secs. Store at room temperature for 10 mins. </li><li>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. </li><li>Option 1: Add 50 µl of <a href="#TE buffer" >TE buffer</a>. (or) Option 2: Add 50 µl of water. Resuspend the pellet by vortexing/by shaking vigorously. You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli. </li></ol></html>

Yeast DNA Prep protocol

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