Ethanol precipitation of small DNA fragments protocol

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Revision as of 23:41, 30 September 2009 by Vaishnavi Ananth (talk | contribs)
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>absolute ethanol stored at -20°C</li><li>DNA sample</li><li>95% ethanol stored at room temperature</li><li>water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out DNA sample into an Eppendorf tube.<br>Add <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute ethanol</font>.<br><font color = "#800517"><i>Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>The long incubation time is critical for small fragments.</i></font><br></li></p><p><li>Centrifuge at a speed of at least <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>30 mins</font></b> at <b><font color=#357EC7>0°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>750 - 1000 µl</font></b> of <font color=#357EC7>95% ethanol</font>.<br><font color = "#800517"><i>Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.</i></font><br></li></p><p><li>Centrifuge at a speed of at least <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>10 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>Dry the pellet in air. <br><font color = "#800517"><i>I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.</i></font><br></li></p><p><li>Add <font color=#357EC7>water</font> to pellet.<br><font color = "#800517"><i>Use appropriate volume of water.</i></font><br>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H<sub>2</sub>O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H<sub>2</sub>O has the advantage of keeping the salt content of your ligation reaction down.</i></font><br></li></p></ol></html>