Team:Copenhagen/Protocols
From 2012.igem.org
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+ | <html> | ||
+ | <table id="graa" cellpadding=20px><td width="660px" height="100%" valign="top" ><p align="justify"> | ||
+ | <a name="1"></a><h2> xxx </h2> | ||
+ | xxx </p> | ||
+ | |||
+ | <a name="2"></a><h2>Protocol for Polymerase Chain Reaction Site-Directed Mutagenesis</h2> | ||
+ | We used PCR SDM to remove a Xbal restriction site from the luxCDABE cassette to make it compatible with the Biobrick system. | ||
+ | <br> | ||
+ | <br> | ||
+ | <b>Primer design</b> | ||
+ | <br> | ||
+ | Two complementary primers of 25-30 basepairs are designed. | ||
+ | The chosen sequence should be identical to the template strand, except for the single basepair which is to be mutated. | ||
+ | The melting temperature of the two primers are noted. | ||
+ | <br> | ||
+ | <br> | ||
+ | <b>PCR reaction</b> | ||
+ | <br> | ||
+ | Each sample reaction is prepared in a PCR-tube as indicated below: | ||
+ | <ul> | ||
+ | <li> X µL of primer 1 for a total of 125 ng of DNA | ||
+ | <li> X µL of primer 2 for a total of 125 ng of DNA | ||
+ | <li> 1 µL of template dsDNA | ||
+ | <li> 10 µL of Phusion buffer (CONC???) | ||
+ | <li> 1 µL of dNTPs (CONC???) | ||
+ | <li>0.5 µL of Phusion Hotstart enzyme (CONC???) | ||
+ | <li> X µL of ddH2O to a total volume of 50 microliters | ||
+ | </ul> | ||
+ | <br> | ||
+ | <b>Notes</b> | ||
+ | <br> | ||
+ | <ul> | ||
+ | <li>The template dsDNA should be complete plasmids obtained an purified from an overnight culture. The length of the plasmid should be known. | ||
+ | <li>A series of template dsDNA dilutions can be included in the range 5-50 ng of dsDNA per reaction. The remaining concentrations should be held constant. | ||
+ | <li>A control reaction containing ddH2O instead of template should be included. | ||
+ | </ul> | ||
+ | |||
+ | <a name="3"></a><h2> xxx </h2> | ||
+ | |||
+ | xxx | ||
+ | |||
+ | <br> | ||
+ | </a> | ||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | <td width="182px" height="100%" valign="top" ><p>Choose protocol</p> | ||
+ | <div id="xmenu"> | ||
+ | |||
+ | <a href="https://2012.igem.org/Team:Copenhagen/Parts#1" class="h2"><b>xxx</b> </a><br> | ||
+ | <a href="https://2012.igem.org/Team:Copenhagen/Parts#2" class="h2"><b>xxx</b> </a><br> | ||
+ | <a href="https://2012.igem.org/Team:Copenhagen/Parts#3" class="h2"><b>xxx</b> </a><br> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </td> | ||
+ | </th> | ||
+ | </table> | ||
+ | </html> |
Revision as of 10:53, 23 September 2012