Yeast DNA Prep protocol: Difference between revisions
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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 <i><br><tab><div style="margin-right: 600px;">(near saturation)</div></i></a></li><li> <a name="breaking buffer">breaking buffer <i><br><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></i></a></li><li> <a name="ice-cold phenol : chloroform : isoamyl alcohol"> ice-cold phenol : chloroform : isoamyl alcohol <i><br><tab><div style="margin-right: 600px;">(25:24:1)</div></i></a></li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0)</div></i></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><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <a href="#yeast culture grown up to appropriate density" ><font color=#357EC7>yeast culture grown up to appropriate density</font></a> into | <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 <i><br><tab><div style="margin-right: 600px;">(near saturation)</div></i></a></li><li> <a name="breaking buffer">breaking buffer <i><br><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></i></a></li><li> <a name="ice-cold phenol : chloroform : isoamyl alcohol"> ice-cold phenol : chloroform : isoamyl alcohol <i><br><tab><div style="margin-right: 600px;">(25:24:1)</div></i></a></li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0)</div></i></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><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <a href="#yeast culture grown up to appropriate density" ><font color=#357EC7>yeast culture grown up to appropriate density</font></a> into Eppendorf tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>1 min</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Measure out <b><font color=#357EC7>200 µl</font></b> of <a href="#breaking buffer" ><font color=#357EC7>breaking buffer</font></a> into Eppendorf tube (1).<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <a href="#ice-cold phenol : chloroform : isoamyl alcohol" ><font color=#357EC7>ice-cold phenol : chloroform : isoamyl alcohol</font></a>.<br>Add <b><font color=#357EC7> 200 mg</font></b> of <font color=#357EC7>glass beads</font>.<br><font color = "#800517"><i>Wear gloves for this step!</i></font><br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>2.5 mins</font></b> .<br><font color = "#800517"><i>Be careful when vortexing; label can be dissolved by the phenol.</i></font><br><font color = "#800517"><i>Hold cap tightly so it doesn't open or spill.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <a href="#TE buffer" ><font color=#357EC7>TE buffer</font></a>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out <b><font color=#357EC7>350 µl</font></b> of top layer.<br>Transfer top aqueous layer into Eppendorf tube (2).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 95% ethanol</font> into Eppendorf tube (2).<br>Vortex the mixture for a few secs.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Stand the tube containing pellet for <b><font color=#357EC7>10 mins</font></b> in an inverted position on a paper towel to allow all of the fluid to drain away.<br></li></p><p><li><p><b>Option 1: </b>Add <b><font color=#357EC7>50 µl</font></b> of <a href="#TE buffer" ><font color=#357EC7>TE buffer</font></a>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>50 µl</font></b> of <font color=#357EC7>water</font>.<br></p><p>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 30 mins</font></b></p></html> | ||
Latest revision as of 06:33, 20 November 2009
<html>
Solutions/reagents:
- <a name="yeast culture grown up to appropriate density">yeast culture grown up to appropriate density
<tab>(near saturation)</a> - <a name="breaking buffer">breaking buffer
<tab>(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)</a> - <a name="ice-cold phenol : chloroform : isoamyl alcohol"> ice-cold phenol : chloroform : isoamyl alcohol
<tab>(25:24:1)</a> - <a name="TE buffer">TE buffer
<tab>(10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0)</a> - ice-cold 95% ethanol
- water
- glass beads
Equipment:
- Centrifuge
- Eppendorf tubes
Steps:
- 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
</html>
