TITLE: E. coli Colony PCR
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Ideally use at least two master mixes:</div><div class = "text-block">2 vector-specific primers (to determine insert size): </div><div class = "text-block">1 vector-specific + 1 gene-specific primer </div><div class = "text-block">To safe resources one of them sould be enough. Is not as neat as it should be but functional. Use Vector + gene specific, because than you can be sure, that the gene is inserted in the right direction</div><div class = "text-block"><span style = "font-weight:bold;">For genotyping of putative colonies</span></div><div class = "text-block">Prepare a PCR master mix for 9 reactions, as follows:</div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td><td>C</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">1x Reaction   (Volume; µL)</td><td rowspan = "" colspan = "" style ="display : table-cell;">9x Reactions   (Volume; µL)</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">Sterile MilliQ Water</td><td rowspan = "" colspan = "" style ="display : table-cell;">8</td><td rowspan = "" colspan = "" style ="display : table-cell;">72</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">Red Taq 2x Master Mix(1.5 mM MgCl2)</td><td rowspan = "" colspan = "" style ="display : table-cell;">10</td><td rowspan = "" colspan = "" style ="display : table-cell;">90</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">F Primer (10 µM)</td><td rowspan = "" colspan = "" style ="display : table-cell;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">9</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">R Primer (10 µM)</td><td rowspan = "" colspan = "" style ="display : table-cell;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">9</td></tr><tr><td style = "text-align:center;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">Total   Volume</td><td rowspan = "" colspan = "" style ="display : table-cell;">20</td><td rowspan = "" colspan = "" style ="display : table-cell;">180</td></tr></table><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Aliquot 20 µL of the master mix in the 8 tubes of a PCR strip.</li><li style = "counter-reset:ol0;list-style-type:disc;">Transfer cells from a single colony (8 in total) using a sterile P2 pipette tip. </li><li style = "counter-reset:ol0;list-style-type:disc;">Run thermocycler using the following program (extension for 1.5 kb insert):</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">StepTemp (°C)Time (mm:ss)Purpose1953:00Initial Denaturation25 cycles of Steps 2 to   4:2950:20Denaturation3570:30Annealing4721:00Extension5725:00Final Extension64∞Storage</span></a><span></span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Analyze bands on a 1% Agarose gel:</li></ul></div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td><td>C</td><td>D</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Step</td><td rowspan = "" colspan = "" style ="display : table-cell;">Temp (°C)</td><td rowspan = "" colspan = "" style ="display : table-cell;">Time (mm:ss)</td><td rowspan = "" colspan = "" style ="display : table-cell;">Purpose</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">95</td><td rowspan = "" colspan = "" style ="display : table-cell;">3:00</td><td rowspan = "" colspan = "" style ="display : table-cell;">Initial Denaturation</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">25 cycles of Steps 2 to   4:</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">95</td><td rowspan = "" colspan = "" style ="display : table-cell;">0:20</td><td rowspan = "" colspan = "" style ="display : table-cell;">Denaturation</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">57</td><td rowspan = "" colspan = "" style ="display : table-cell;">0:30</td><td rowspan = "" colspan = "" style ="display : table-cell;">Annealing</td></tr><tr><td style = "text-align:center;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">72</td><td rowspan = "" colspan = "" style ="display : table-cell;">1:00</td><td rowspan = "" colspan = "" style ="display : table-cell;">Extension</td></tr><tr><td style = "text-align:center;">7</td><td rowspan = "" colspan = "" style ="display : table-cell;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">72</td><td rowspan = "" colspan = "" style ="display : table-cell;">5:00</td><td rowspan = "" colspan = "" style ="display : table-cell;">Final Extension</td></tr><tr><td style = "text-align:center;">8</td><td rowspan = "" colspan = "" style ="display : table-cell;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">∞</td><td rowspan = "" colspan = "" style ="display : table-cell;">Storage</td></tr></table><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block">(no amplification if Gene is missing or in wrong direction when using gene specific primer)</div><div class = "text-block">(Smaller than expected band if no insert is present when using two vector specific primer)</div></div>

[STEPS]