Mouse tissue lysis for genotyping protocol: Difference between revisions
(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="Taq DNA polymerase 10X buffer with MgCl<sub>2</sub>">Taq DNA polymerase 10X buffer with MgCl<sub>2</sub> <i><br><tab><div...) |
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Latest revision as of 04:35, 22 March 2010
<html> <h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="Taq DNA polymerase 10X buffer with MgCl<sub>2</sub>">Taq DNA polymerase 10X buffer with MgCl<sub>2</sub> <i><br><tab><div style="margin-right: 600px;">(diluted 1:10 in ddH<sub>2</sub>)</div></i></a></li><li> <a name="proteinase K">proteinase K <i><br><tab><div style="margin-right: 600px;">(20 mg/ml)</div></i></a></li><li>tail or tissue chunk</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Tissue lysis to release DNA</font></b><br>Measure out <b><font color=#357EC7>100 µl</font></b> of <a href="#Taq DNA polymerase 10X buffer with MgCl<sub>2</sub>" ><font color=#357EC7>Taq DNA polymerase 10X buffer with MgCl<sub>2</sub></font></a> into tail or tissue chunk.<br>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#proteinase K" ><font color=#357EC7>proteinase K</font></a>.<br></li></p><p><li>Incubate at <b><font color=#357EC7>50°C</font></b> for <b><font color=#357EC7>3 - 12 hrs(overnight)</font></b> with shaking at 300 rpm.<br><font color = "#800517"><i>proteinase K is stable over a broad pH range (4.0 to 12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65°C.</i></font><br></li></p><p><li><b><font size=3>Proteinase K inactivation</font></b><br>Incubate at <b><font color=#357EC7>95°C</font></b> for <b><font color=#357EC7>10 - 20 mins</font></b> with shaking at 300 rpm.<br>Centrifuge at a speed of <font color=#357EC7>13000 rpm</font> for <b><font color=#357EC7>3 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out <b><font color=#357EC7>80 µl</font></b> of top layer into sterile 1.5-ml microcentrifuge tube (2).<br>Discard bottom layer.<br></li></p><p><li><b><font size=3>Storage before PCR</font></b><br>NOTE: If sample is/are used within <b><font color=#357EC7>1 hr</font></b>, set temperature to <b><font color=#357EC7>4°C</font></b> and use, else store it at <b><font color=#357EC7>-20°C</font></b>.</li></p></ol> </html>
