Knight:Colony PCR protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR supermix</li><li> <a name="VF2">VF2 <tab><div style="margin-right: 600px;">(40 µM)</div></a></li><li> <a name="VR">VR <tab><div style="margin-right: 600px;">(40 µM)</div></a></li><li>colony template</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Electrophoretic unit</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><li>Reaction Mixture Use the following table as a checklist for preparing the reaction in reaction tube (1): <table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td>PCR supermix</td><td>VF2</td><td>VR</td><td>colony template</td></tr></thead><tbody><tr><td>Colony PCR</td><td>9 µl</td><td>0.25 µl</td><td>0.25 µl</td><td>0.5 µl</td></tr></body></table></li><li>PCR conditions Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation <ul><li>Denature: 95°C, 15 mins</li></ul>Thermocycling <ul><li>No. of cycles: 39</li><li>Denature: 94°C, 30 secs</li> <li> Anneal: 56°C, 30 secs</li> <li>Elongate: 68°C, 60 secs</li></ul>Elongation time : 1 min per kb of expected product. 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. Termination<ul><li>Elongate: 60°C, 20 mins</li><li>Hold: 4°C, until removed from machine </li></ul></li><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. </li></ol>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 55 mins</html>

Knight:Colony PCR protocol

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