Chromosomal DNA isolation from E. coli protocol: Difference between revisions
(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>culture grown in your favorite medium</li><li>ice-cold Killing Buffer</li><li>TE</li><li>10% SDS</li><li>0.5M EDTA</li><li>isopropa...) |
(No difference)
|
Revision as of 23:05, 30 September 2009
<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>culture grown in your favorite medium</li><li>ice-cold Killing Buffer</li><li>TE</li><li>10% SDS</li><li>0.5M EDTA</li><li>isopropanol</li><li> <a name="RNase A">RNase A <i><br><tab><div style="margin-right: 600px;">(25mg/ml)</div></i></a></li><li> <a name="proteinase K">proteinase K <i><br><tab><div style="margin-right: 600px;">(25mg/ml)</div></i></a></li><li>ethanol</li><li>3M Na-Acetate</li><li>70% ethanol</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out culture grown in your favorite medium into an Eppendorf tube.<br>Add <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold Killing Buffer</font>.<br>Vortex the mixture for a few secs.<br>Store the tube on ice.<br><font color = "#800517"><i>Samples should be processed as fast as possible.</i></font><br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>TE</font>.<br>Add <b><font color=#357EC7>40 µl</font></b> of <font color=#357EC7>10% SDS</font>.<br>Add <b><font color=#357EC7>3 µl</font></b> of <font color=#357EC7>0.5M EDTA</font>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>isopropanol</font>.<br>Vortex the mixture for a few secs.<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>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>TE</font>.<br>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#RNase A" ><font color=#357EC7>RNase A</font></a>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#proteinase K" ><font color=#357EC7>proteinase K</font></a>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li><font color = "red"><i>phenol extract (2x phenol & 2x chlorophorm).</i></font><br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ethanol</font>.<br>Add <b><font color=#357EC7>40 µl</font></b> of <font color=#357EC7>3M Na-Acetate</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<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>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>50 µl</font></b> of <font color=#357EC7>distilled water</font>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p></ol></html>
