DNA extraction from tissue protocol

<html> <h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="DNA extraction buffer">DNA extraction buffer <tab><div style="margin-right: 600px;">(98µl ReagentB + 2µl ProteinaseK, mix fresh)</div></a></li><li>24:1 Chloroform:Isoamyl alcohol</li><li> <a name="PCI mix">PCI mix <tab><div style="margin-right: 600px;">(One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol, shake thoroughly to make emulsion)</div></a></li><li>100% EtOH</li><li>3M sodium acetate pH 5.0</li><li>70% EtOH</li><li>MilliQ water</li><li>small piece of tissue or embryo</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>Proteinase K digestion <ol type="a"><li>Measure out 100 µl of <a href="#DNA extraction buffer" >DNA extraction buffer</a> into sterile 1.5-ml microcentrifuge tube (1). 100 µl is enough for a small pea size chunk of tissue or one embryo. </li><li>Add small piece of tissue or embryo. </li><li>Incubate at 50°C for 12 hrs(overnight). </li></ol></li><li>Phenol/chloroform/isoamyl (PCI) <ol type="a"><li>Add 1 volume <a href="#PCI mix" >PCI mix</a>. </li><li>Vortex the mixture for 10 secs . </li><li>Centrifuge at maximum speed for 5 mins at room temperature 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><li>Add 1 volume 24:1 Chloroform:Isoamyl alcohol to sterile 1.5-ml microcentrifuge tube (2). </li><li>Vortex the mixture for 10 secs . </li><li>Centrifuge at maximum speed for 5 mins at room temperature 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 <ol type="a"><li>Add 2 volumes 100% EtOH to sterile 1.5-ml microcentrifuge tube (3). </li><li>Add 0.1 volume 3M sodium acetate pH 5.0. </li><li>Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 150 µl of 70% EtOH. </li><li>Centrifuge at maximum speed for 2 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Dry the pellet in air. </li><li>Add MilliQ water to pellet. Resuspend pellet by vortexing/by shaking vigorously. </li></ol>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 22 mins </html>

DNA extraction from tissue protocol

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