DNA extraction from tissue protocol

Solutions/reagents:  DNA extraction buffer  (98µl ReagentB + 2µl ProteinaseK, mix fresh) 24:1 Chloroform:Isoamyl alcohol PCI mix  (One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol, shake thoroughly to make emulsion) 100% EtOH3M sodium acetate pH 5.070% EtOHMilliQ water</li>small piece of tissue or embryo</li></ul> Equipment: Incubator</li>Centrifuge</li>Sterile 1.5-ml microcentrifuge tubes</li></ul> Steps: <ol>  Proteinase K digestion  Measure out <font color=#357EC7>100 µl  of <a href="#DNA extraction buffer" ><font color=#357EC7>DNA extraction buffer </a> into sterile 1.5-ml microcentrifuge tube (1). <font color = "#800517">100 µl is enough for a small pea size chunk of tissue or one embryo. </li> Add <font color=#357EC7>small piece of tissue or embryo. </li> Incubate at <font color=#357EC7>50°C  for <font color=#357EC7>12 hrs (overnight). </li> </ol></li>  Phenol/chloroform/isoamyl (PCI)  Add <font color=#357EC7>1  volume <a href="#PCI mix" ><font color=#357EC7>PCI mix </a>. </li> Vortex the mixture for <font color=#357EC7>10 secs . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2). Discard bottom layer. Repeat this step as needed. </li> Add  <font color=#357EC7>1  volume <font color=#357EC7>24:1 Chloroform:Isoamyl alcohol to sterile 1.5-ml microcentrifuge tube (2). </li> Vortex the mixture for <font color=#357EC7>10 secs . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> 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> Ethanol precipitation  <li>Add  <font color=#357EC7>2  volumes <font color=#357EC7>100% EtOH to sterile 1.5-ml microcentrifuge tube (3). </li> <li>Add  <font color=#357EC7>0.1  volume <font color=#357EC7>3M sodium acetate pH 5.0. </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>10 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> <li>Add <font color=#357EC7>150 µl  of <font color=#357EC7>70% EtOH. </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>2 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> <li>Dry the pellet in air. </li> <li>Add <font color=#357EC7>MilliQ water to pellet. Resuspend pellet by vortexing/by shaking vigorously. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 12 hrs, 22 mins