DNA extraction - Salting Out protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="Digestion Buffer">Digestion Buffer <i><br><tab><div style="margin-right: 600px;">(10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS)</div></i></a></li><li> <a name="Proteinase K">Proteinase K <i><br><tab><div style="margin-right: 600px;">(20mg/ml)</div></i></a></li><li> <a name="Sodium Acetate pH 5.2">Sodium Acetate pH 5.2 <i><br><tab><div style="margin-right: 600px;">(3M)</div></i></a></li><li>ice-cold 98% ethanol</li><li>ice-cold 70% ethanol</li><li>1X TE</li><li>water</li><li>tissue</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Tissue Digestion</font></b><br><ol type="a"><p><li>Measure out <a href="#Digestion Buffer" ><font color=#357EC7>Digestion Buffer</font></a> into sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>0.005</font></b> volume <a href="#Proteinase K" ><font color=#357EC7>Proteinase K</font></a>.<br><font color = "#800517"><i>That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK.</i></font><br></li></p><p><li>Homogenize tissue in solution.<br></li></p><p><li>Incubate at <b><font color=#357EC7>55°C</font></b> for <b><font color=#357EC7>1 - 12 hrs(overnight)</font></b>.<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>2 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Precipitation of Protein and Cell Debris</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>0.1</font></b> volume <a href="#Sodium Acetate pH 5.2" ><font color=#357EC7>Sodium Acetate pH 5.2</font></a> to sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Incubate at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>20 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (4).<br>Discard bottom layer.<br><font color = "#800517"><i>Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube.</i></font><br></li></p></ol></li></p><p><li><b><font size=3>Precipitation of Nucleic Acids</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>ice-cold 98% ethanol</font> to sterile 1.5-ml microcentrifuge tube (4).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Incubate at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>20 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>1 ml</font></b> of <font color=#357EC7>ice-cold 98% ethanol</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>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 70% ethanol</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>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p></ol></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 70% ethanol</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>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br><p><b>Option 1: </b>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>1X TE</font>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>water</font>.<br></p><p>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Ensure to dry the pelletted DNA completely before attempting to resuspend.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 27 mins</font></b></p></html>