Small Scale Plasmid Isolation (Maxiprep) protocol

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Revision as of 00:53, 23 October 2009 by Vaishnavi Ananth (talk | contribs) (New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RN...)
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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Centrifuge bottles</li><li>Sterile 15-ml centrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>50 ml</font></b> of <font color=#357EC7>overnight culture</font> into a centrifuge bottle.<br>Centrifuge at <font color=#357EC7>maximum speed</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></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold Solution I</font>.<br>Resuspend ice-cold Solution I by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>Solution II</font>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>Solution III</font>.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</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>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 15-ml centrifuge tube (1).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Phenol/Chloroform Cleanup</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font> to sterile 15-ml centrifuge tube (1).<br>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br></li></p><p><li>Vortex the mixture for a few secs.<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 the top layer.<br>Transfer top aqueous layer into sterile 15-ml centrifuge tube (2).<br>Discard bottom layer.<br></li></p></ol></li></p><p><b><font size=3>RNase (Optional)</font></b><br><ol type="a"><p><li>Measure out RNase to a final concentration of 3µg/ml into sterile 15-ml centrifuge tube (2).<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 - 45 mins</font></b>.<br><font color = "#800517"><i>Use a water bath.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font>.<br>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br>Vortex the mixture for a few secs.<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 the top layer.<br>Transfer top aqueous layer into sterile 15-ml centrifuge tube (3).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Alcohol Precipitation / Purification</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold isopropanol</font> to sterile 15-ml centrifuge tube (3).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</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>15 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Mix solution by pipetting up and down several times.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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></li></p><p><li>Dry the pellet in air for at most <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>200 - 300 µl</font></b> of <font color=#357EC7>sterile distilled water</font>.<br>Resuspend sterile distilled water by vortexing/by shaking vigorously.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol> </html>