Richard Lab:Restriction Digest protocol: Difference between revisions
(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="prepared DNA">prepared DNA <i><br><tab><div style="margin-right: 600px;">(from Miniprep, PCR or Gel Extraction)</div></i><...) |
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Revision as of 22:01, 20 October 2009
<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="prepared DNA">prepared DNA <i><br><tab><div style="margin-right: 600px;">(from Miniprep, PCR or Gel Extraction)</div></i></a></li><li>restriction endonucleases</li><li>10X restriction endonuclease buffer</li><li> <a name="BSA">BSA <i><br><tab><div style="margin-right: 600px;">(optional)</div></i></a></li><li>phosphatase</li><li>phosphatase buffer</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><font color = "red"><i>Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.</i></font><br></li></p><p><li>Measure out <a href="#prepared DNA" ><font color=#357EC7>prepared DNA</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br>Add <b><font color=#357EC7>4</font></b> volumes <font color=#357EC7>distilled water</font>.<br>Use the following table as a checklist for preparing the reaction in sterile 1.5-ml microcentrifuge tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td><font color=#357EC7>10X restriction endonuclease buffer</font></td><td><font color=#357EC7>BSA</font></td><td><font color=#357EC7>DNA solution</font></td><td><font color=#357EC7>restriction endonucleases</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digestion</font></td><td><b><b><font color=#357EC7>5 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>40 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Use a water bath for incubation.</i></font><br></li></p><p><li>Perform enzyme inactivation by storing at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li><b><font size=2>If digesting vector:</font></b><br>Measure out <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>phosphatase</font> into sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>5 µl</font></b> of <font color=#357EC7>phosphatase buffer</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>45 mins</font></b>.<br>Perform enzyme inactivation by storing at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol></html>
