DNA extraction - Salting Out protocol

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

DNA extraction - Salting Out protocol

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