Ethanol precipitation of small DNA fragments protocol

From OpenWetWare
Jump to navigationJump to search

<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></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute ethanol</font> into DNA sample.<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>Measure out <b><font color=#357EC7>750 - 1000 µl</font></b> of <font color=#357EC7>95% ethanol</font> into Eppendorf tube (1).<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 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 H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 40 mins</font></b></p></html>