One step 'miniprep' method for the isolation of plasmid DNA protocol

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Revision as of 01:27, 29 September 2009 by Vaishnavi Ananth (talk | contribs) (New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight E.coli culture</li><li> <a name="phenol : chloroform : isoamyl alcohol(25:24:1)">phenol : chloroform : isoamyl alcohol(25...)
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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight E.coli culture</li><li> <a name="phenol : chloroform : isoamyl alcohol(25:24:1)">phenol : chloroform : isoamyl alcohol(25:24:1) <i><br><tab><div style="margin-right: 600px;">(phenol saturated with TE(10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol)</div></i></a></li><li>isopropanol</li><li>70% ethanol</li><li>100 µl/ml RNAse</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Flasks of appropriate volumes</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>overnight E.coli culture</font> into a sterile 1.5-ml microcentrifuge tube.<br><font color = "#800517"><i>We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5 ml</font></b> of <a href="#phenol : chloroform : isoamyl alcohol(25:24:1)" ><font color=#357EC7>phenol : chloroform : isoamyl alcohol(25:24:1)</font></a>.<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>1 min</font></b> .<br><font color = "#800517"><i>Vortex at maximum speed.</i></font><br><font color = "#800517"><i>Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>12000 Xg</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>.<br></li></p><p><li><b>Meanwhile:</b><br>Set aside a fresh a sterile 1.5-ml microcentrifuge tube. Call it Tube I. <br>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>isopropanol</font> into Tube I.<br></li></p><p><li>Measure out <b><font color=#357EC7>0.45 ml</font></b> of <font color=#357EC7>top aqueous phase obtained after centrifugation</font> into Tube I.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>12000 Xg</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br><font color = "#800517"><i>Addition of salt and cooling is unnecessary.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br><font color = "#800517"><i>Add ethanol carefully to the side of the tube.</i></font><br>Discard solution.<br></li></p><p><li>Repeat Step 7. <br>Add <font color=#357EC7>100 µl/ml RNAse</font> to solution.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.</i></font><br></li></p></ol> </html>