DNA extraction - Salting Out protocol

Solutions/reagents:  Digestion Buffer  (10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS)  Proteinase K  (20mg/ml)  Sodium Acetate pH 5.2  (3M) ice-cold 98% ethanolice-cold 70% ethanol1X TE</li>water</li>tissue</li></ul> Equipment: Incubator</li>Centrifuge</li>Sterile 1.5-ml microcentrifuge tubes</li></ul> Steps: <ol>  Tissue Digestion  Measure out <a href="#Digestion Buffer" ><font color=#357EC7>Digestion Buffer </a> into sterile 1.5-ml microcentrifuge tube (2). Add <font color=#357EC7>0.005  volume <a href="#Proteinase K" ><font color=#357EC7>Proteinase K </a>. <font color = "#800517">That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK. </li> Homogenize tissue in solution. </li> Incubate at <font color=#357EC7>55°C  for <font color=#357EC7>1 - 12 hrs(overnight) . </li> Vortex the mixture for a few secs. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>2 mins  at <font color=#357EC7>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>  Precipitation of Protein and Cell Debris  Add <font color=#357EC7>0.1  volume <a href="#Sodium Acetate pH 5.2" ><font color=#357EC7>Sodium Acetate pH 5.2 </a> to sterile 1.5-ml microcentrifuge tube (3). </li> Close the tube tightly and gently mix the contents by inverting the tube. Incubate at <font color=#357EC7>-20°C  for <font color=#357EC7>15 mins . Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>20 mins  at <font color=#357EC7>4°C  and aspirate out the top layer. Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (4). Discard bottom layer. <font color = "#800517">Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube. </li> </ol></li>  Precipitation of Nucleic Acids  Add  <font color=#357EC7>2  volumes <font color=#357EC7>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 <font color=#357EC7>-20°C  for <font color=#357EC7>15 mins . </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>20 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. </li> <li>Add <font color=#357EC7>1 ml  of <font color=#357EC7>ice-cold 98% ethanol. Vortex the mixture for a few secs. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>1 ml  of <font color=#357EC7>ice-cold 70% ethanol. Vortex the mixture for a few secs. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. </li> </ol></li>  (Optional)  Add <font color=#357EC7>1 ml  of <font color=#357EC7>ice-cold 70% ethanol. Vortex the mixture for a few secs. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. </li> <li>Dry the pellet in air. Option 1: Add <font color=#357EC7>10 µl  of <font color=#357EC7>1X TE. (or) Option 2: Add <font color=#357EC7>10 µl  of <font color=#357EC7>water. Resuspend pellet by vortexing/by shaking vigorously. <font color = "#800517">Ensure to dry the pelletted DNA completely before attempting to resuspend. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 27 mins