Knight:Colony PCR protocol

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Revision as of 02:08, 24 September 2009 by Vaishnavi Ananth (talk | contribs) (New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR supermix</li><li> <a name="VF2">VF2 <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li> <a name="VR">V...)
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR supermix</li><li> <a name="VF2">VF2 <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li> <a name="VR">VR <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li>colony template</li></ul><h2>Parameters:</h2><ul type="circle"><li>X - 1 min per kb of expected product.</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Electrophoretic unit</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Reaction Mixture</font></b><br>Use the following table as a checklist for preparing the reaction:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>PCR supermix</font></td><td><font color=#357EC7>VF2</font></td><td><font color=#357EC7>VR</font></td><td><font color=#357EC7>colony template</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Colony PCR</font></td><td><b><b><font color=#357EC7>9 µl</font></b></td><td><b><b><font color=#357EC7>0.25 µl</font></b></td><td><b><b><font color=#357EC7>0.25 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></td></tr></body></table></li></p><p><li><b><font size=3>PCR conditions</font></b><br>Program a standard thermocycler to run the reaction using the following parameters:<br>Initial denaturation<br><ul><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>15 mins</font></b></li></ul>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>39</font></b></li><li>Denature: <b><font color=#357EC7>94°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li> Anneal: <b><font color=#357EC7>56°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li>Elongate: <b><font color=#357EC7>68°C</font></b>, <b><font color=#357EC7>X</font></b></li></ul><font color = "#800517"><i>Elongation time : I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.</i></font><br>Termination<ul><li>Elongate: <b><font color=#357EC7>60°C</font></b>, <b><font color=#357EC7>20 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul></li></p><p><li>Perform agarose gel electrophoresis of appropriate quantity of PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.<br></li></p></ol></html>