Yeast DNA Prep protocol

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</li>water</li>glass beads</li></ul> Equipment: Centrifuge</li>Eppendorf tubes</li></ul> Steps: <ol> Measure out <font color=#357EC7>1.5 ml  of <a href="#yeast culture grown up to appropriate density" ><font color=#357EC7>yeast culture grown up to appropriate density </a> into Eppendorf tube (1). Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>1 min  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> Measure out <font color=#357EC7>200 µl  of <a href="#breaking buffer" ><font color=#357EC7>breaking buffer </a> into Eppendorf tube (1). Resuspend pellet by vortexing/by shaking vigorously. </li> Add <font color=#357EC7>200 µl  of <a href="#ice-cold phenol : chloroform : isoamyl alcohol" ><font color=#357EC7>ice-cold phenol : chloroform : isoamyl alcohol </a>. Add <font color=#357EC7> 200 mg  of <font color=#357EC7>glass beads. <font color = "#800517">Wear gloves for this step! </li> Vortex the mixture for <font color=#357EC7>2.5 mins . <font color = "#800517">Be careful when vortexing; label can be dissolved by the phenol. <font color = "#800517">Hold cap tightly so it doesn't open or spill. </li> Add <font color=#357EC7>200 µl  of <a href="#TE buffer" ><font color=#357EC7>TE buffer </a>. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out <font color=#357EC7>350 µl  of top layer. Transfer top aqueous layer into Eppendorf tube (2). Discard bottom layer. </li> Measure out <font color=#357EC7>1 ml  of <font color=#357EC7>ice-cold 95% ethanol into Eppendorf tube (2). Vortex the mixture for a few secs. Store at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>10 mins . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>2 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. Stand the tube containing pellet for <font color=#357EC7>10 mins  in an inverted position on a paper towel to allow all of the fluid to drain away. </li>  Option 1: Add <font color=#357EC7>50 µl  of <a href="#TE buffer" ><font color=#357EC7>TE buffer </a>. (or) Option 2: Add <font color=#357EC7>50 µl  of <font color=#357EC7>water. Resuspend pellet by vortexing/by shaking vigorously. <font color = "#800517">You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 30 mins