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<h1>Protocols: <small>Use the categories on the left to find the protocol you need:</small></h1>
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<div id="one">&nbsp;</div>
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<div id="three"><h3>Protocols</h3></div>
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    <div id="two">&nbsp;</div>
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<h1><small>Select one of the categories below to find the protocol you need:</small></h1>
  
 
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                     <li><a href="#a" data-toggle="tab"><strong>PCR</strong></a></li>
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                     <li style="text-align: left;"><a href="#a" data-toggle="tab"><strong>Genetic Tool Development</strong></a></li>
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                     <li class="active"><a href="#b" data-toggle="tab">Two</a></li>
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                     <li style="text-align: center;"><a href="#b" data-toggle="tab"><strong>Imidacloprid Modification</strong></a></li>
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                     <li><a href="#c" data-toggle="tab">Twee</a></li>
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                     <li style="text-align: right;"><a href="#c" data-toggle="tab"><strong>6-CNA Degradation</strong></a></li>
 
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      <p><strong>PCR&nbsp;</strong><br />
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<br />
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The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1). <em>Taq</em> DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using NEB's <em>Taq</em> DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization.</p>
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<h2>Protocol </h2>
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<p><strong>Reaction setup:</strong>&nbsp;<br />
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<br />
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We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95&deg;C).&nbsp;<d>
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</d>
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<table>
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    <tbody>
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        <tr>
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            <td><strong>Component</strong></td>
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            <td><strong>25 &mu;l reaction</strong></td>
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            <td><strong>50 &mu;l reaction</strong></td>
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            <td><strong>Final Concentration</strong></td>
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        </tr>
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        <tr>
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            <td valign="top">10X Standard <em>Taq </em>Reaction Buffer </td>
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            <td valign="top">2.5 &mu;l </td>
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            <td valign="top">5 &mu;l </td>
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            <td valign="top">1X </td>
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        </tr>
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        <tr>
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            <td>10 mM dNTPs</td>
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            <td>0.5 &micro;l</td>
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            <td>1 &mu;l</td>
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            <td>200 &micro;M</td>
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        </tr>
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        <tr>
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            <td>10 &micro;M Forward Primer</td>
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            <td>0.5 &micro;l</td>
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            <td>1 &mu;l</td>
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            <td>0.2 &micro;M (0.05&ndash;1 &micro;M)</td>
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        </tr>
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        <tr>
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            <td>10 &micro;M Reverse Primer</td>
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            <td>0.5 &micro;l</td>
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            <td>1 &mu;l</td>
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            <td>0.2 &micro;M (0.05&ndash;1 &micro;M)</td>
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        </tr>
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        <tr>
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            <td>Template DNA</td>
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            <td>variable</td>
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            <td>variable</td>
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            <td>&lt;1,000 ng</td>
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        </tr>
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        <tr>
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            <td><em>Taq </em>DNA Polymerase</td>
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            <td>0.125 &micro;l</td>
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            <td>0.25 &micro;l</td>
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            <td>1.25 units/50 &micro;l PCR</td>
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        </tr>
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        <tr>
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            <td>Nuclease-free water</td>
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            <td>to 25 &micro;l</td>
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            <td>to 50 &micro;l</td>
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            <td>&nbsp;</td>
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        </tr>
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    </tbody>
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</table>
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Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.<br />
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<br />
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Transfer PCR tubes from ice to a PCR machine with&nbsp;the block preheated to 95&deg;C and begin thermocycling. <br />
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<br />
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<strong>Thermocycling conditions for a routine PCR:&nbsp;</strong><br />
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<br />
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<table width="309" height="162">
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    <tbody>
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        <tr>
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            <td align="center"><strong>STEP <br />
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            </strong></td>
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            <td align="center"><strong>TEMP<br />
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            </strong></td>
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            <td align="center"><strong>TIME <br />
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            </strong></td>
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        </tr>
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        <tr>
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            <td>Initial Denaturation <br />
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            </td>
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            <td align="center">95&deg;C <br />
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            </td>
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            <td>30 seconds </td>
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        </tr>
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        <tr>
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            <td>30 Cycles </td>
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            <td align="center">95&deg;C<br />
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            45-68&deg;C<br />
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            68&deg;C </td>
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            <td>15-30 seconds<br />
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            15-60 seconds<br />
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            1 minute/kb </td>
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        </tr>
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        <tr>
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            <td>Final Extension </td>
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            <td align="center">68&deg;C </td>
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            <td>5 minutes </td>
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        </tr>
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        <tr>
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            <td>Hold </td>
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            <td align="center">4-10&deg;C </td>
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            <td>&nbsp;</td>
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        </tr>
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    </tbody>
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</table>
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<strong><br />
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General Guidelines:</strong>&nbsp;</p>
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<ol>
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    <li>
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    Template:&nbsp;<br />
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    <br />
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    Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 &mu;l reaction are as follows:&nbsp;
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    <table>
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        <tbody>
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            <tr>
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                <td><strong>DNA</strong></td>
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                <td><strong>Amount</strong></td>
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            </tr>
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            <tr>
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                <td>genomic</td>
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                <td>1 ng&ndash;1 &mu;g</td>
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            </tr>
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            <tr>
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                <td>plasmid or viral</td>
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                <td>1 pg&ndash;1 ng</td>
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            </tr>
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        </tbody>
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    </table>
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    </li>
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    <li>Primers:&nbsp;<br />
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    <br />
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    Oligonucleotide primers are generally 20&ndash;40 nucleotides in length and ideally have a GC content of 40&ndash;60%. Computer programs such as Primer3 (<a re_target="_blank" target="_blank" href="http://frodo.wi.mit.edu/primer3">http://frodo.wi.mit.edu/primer3</a>) can be used to design or analyze primers. The final concentration of each primer in a reaction may be 0.05&ndash;1 &mu;M, typically 0.1&ndash;0.5&nbsp;&mu;M.<br />
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    <br />
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    </li>
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    <li>Mg<sup>++ </sup>and additives:&nbsp;<br />
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    <br />
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    Mg<sup>++ </sup>concentration of 1.5&ndash;2.0 mM is optimal for most PCR products generated with <em>Taq </em>DNA Polymerase. The final Mg<sup>++</sup> concentration in 1X Standard <em>Taq </em>Reaction Buffer is 1.5 mM. This supports satisfactory amplification of most amplicons. However, Mg<sup>++</sup> can be further optimized in 0.5 or 1.0 mM increments using MgCl<sub>2</sub>.&nbsp;<br />
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    <br />
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    Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4).<br />
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    <br />
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    </li>
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    <li>Deoxynucleotides:<br />
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    <br />
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    The final concentration of dNTPs is typically 200 &mu;M of each deoxynucleotide.<br />
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    <br />
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    </li>
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    <li><em>Taq </em>DNA Polymerase Concentration:&nbsp;<br />
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    <br />
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    We generally recommend using <em>Taq</em> DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 &mu;l reaction). However, the optimal concentration of <em>Taq</em> DNA Polymerase may range from 5&ndash;50 units/ml (0.25&ndash;2.5&nbsp;units/50 &mu;l reaction) in specialized applications.<br />
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    <br />
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    </li>
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    <li>Denaturation:&nbsp;<br />
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    <br />
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    An initial denaturation of 30 seconds at 95&deg;C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2&ndash;4 minutes at 95&deg;C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95&deg;C is recommended.&nbsp;<br />
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    <br />
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    During thermocycling a 15&ndash;30 second denaturation at 95&deg;C is recommended.<br />
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    <br />
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    </li>
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    <li>Annealing:&nbsp;<br />
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    <br />
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    The annealing step is typically 15&ndash;60 seconds. Annealing temperature is based on the T<sub>m</sub> of the primer pair and is typically 45&ndash;68&deg;C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5&deg;C below the calculated T<sub>m</sub>.&nbsp; The NEB <a target="_blank" href="/tools-and-resources/interactive-tools/tm-calculator">Tm Calculator </a>is recommended to calculate an appropriate annealing temperature.<br />
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    <br />
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    When primers with annealing temperatures above 65&deg;C are used, a 2-step PCR protocol is possible (see #10).&nbsp;<br />
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    <br />
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    </li>
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−
    <li>Extension:&nbsp;<br />
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    <br />
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−
    The recommended extension temperature is 68&deg;C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68&deg;C is recommended.<br />
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    <br />
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    </li>
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    <li>Cycle number:&nbsp;<br />
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    <br />
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−
    Generally, 25&ndash;35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.<br />
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    <br />
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    </li>
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    <li>2-step PCR:&nbsp;<br />
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    <br />
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−
    When primers with annealing temperatures above 65&deg;C are used, a 2-step thermocycling protocol is possible.<br />
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    <br />
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    <strong>Thermocycling conditions for a routine 2-step PCR:&nbsp;</strong><br />
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    <br />
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    <table>
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        <tbody>
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            <tr>
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                <td align="center"><strong>STEP<br />
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                </strong></td>
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                <td align="center"><strong>TEMP<br />
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                </strong></td>
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                <td align="center"><strong>TIME <br />
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                </strong></td>
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            </tr>
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            <tr>
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                <td>Initial Denaturation<br />
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                </td>
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                <td align="center">95&deg;C<br />
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                </td>
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                <td>30 seconds<br />
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                </td>
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            </tr>
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            <tr>
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                <td>30 Cycles<br />
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                </td>
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                <td align="center">95&deg;C<br />
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                65-68&deg;C<br />
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                </td>
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                <td>15-30 seconds<br />
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                1 minute/kb<br />
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                </td>
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−
            </tr>
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            <tr>
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                <td>Final Extension<br />
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                </td>
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                <td align="center">65-68&deg;C<br />
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                </td>
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                <td>5 minutes<br />
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                </td>
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            </tr>
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            <tr>
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                <td>Hold<br />
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                </td>
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                <td align="center">4-10&deg;C <br />
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                </td>
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                <td>&nbsp;</td>
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            </tr>
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        </tbody>
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    </table>
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    </li>
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−
    <li>PCR product:&nbsp;<br />
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−
    <br />
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−
    The PCR products generated using <em>Taq</em> DNA Polymerase contain dA overhangs at the 3&acute;&ndash;end; therefore the PCR products can be ligated to dT/dU-overhang vectors.</li>
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−
</ol>
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−
<p>References:<br />
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1. <span>Saiki R.K. et al. (1985). <span id="scPublicationTitle_lText" class="bodycopyspecialcharacteroneline"><em>Science</em></span>. 230, 1350-1354.</span><br />
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2. <span>Powell, L.M. et al. (1987). <span id="scPublicationTitle_lText" class="bodycopyspecialcharacteroneline"><em>Cell</em></span>. 50, 831-840.<br />
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3. &nbsp;Sun, Y., Hegamyer, G. and Colburn, N.  (1993). <span class="bodycopyspecialcharacteroneline" id="scPublicationTitle_lText"><em>Biotechniques</em></span>. 15, 372-374.<br />
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4.&nbsp; Sarkar, G., Kapelner, S. and Sommer, S.S.  (1990). <span class="bodycopyspecialcharacteroneline" id="scPublicationTitle_lText"><em>Nucleic
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Acids Res.</em></span>. 18, 7465.</span></p></p>
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</section>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/1/15/UBC_growinggapicolaprotocol.pdf"><h4>Growing<br /> <i>G. apicola</i></h4></a>
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 +
<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/1/1f/UBC_colonyPCRprotocol.pdf"><h4>Colony PCR Amplification</h4></a>
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<div class="tab-pane" id="b">Secondo sed ac orci quis tortor imperdiet venenatis. Duis elementum auctor accumsan. Aliquam in felis sit amet augue.</div>
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<a href="#"><div class="hexagon"><a href="https://static.igem.org/mediawiki/2015/3/3b/UBC_compcellsprotocol.pdf"><h4>Inducing Competence</h4></a>
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</div></a>
  
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<a href="#"><div class="hexagon"><a href="https://static.igem.org/mediawiki/2015/1/1d/UBC_electroporationprotocol.pdf"><h4 style="font-size:16px;">Electroporation</h4></a>
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<a href="#"><div class="hexagon"><a href="https://static.igem.org/mediawiki/2015/7/71/UBC_heatshockprotocol.pdf"><h4>Heat Shock</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/b/b9/Electroporation_IMI_UBC.pdf"><h4>Electroporation</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/6/61/Gibson_Assembly_UBC.pdf"><h4>Gibson Assembly</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/5/5d/Heat_Shock_IMI_UBC.pdf"><h4>Heat Shock</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/a/a5/HPLC_UBC.pdf"><h4>HPLC</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/7/7f/Making_Megaprimers_UBC.pdf"><h4>Making Megaprimers</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/f/fc/Mutagenesis_PCR_UBC.pdf"><h4>Mutagenesis PCR</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/c/c7/SDS_Page_IMI_UBC.pdf"><h4>SDS Page</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/a/aa/Sonication_UBC.pdf"><h4>Sonication</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/5/56/Whole_Plasmid_PCR_UBC.pdf"><h4>Whole Plasmid PCR</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/2/2f/Restriction_Digest_UBC.pdf"><h4>Restriction Digest</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/6/68/GCMS_Protocol_UBC.pdf"><h4>GC/MS Conditions & Preparation</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/2/2f/Restriction_Digest_UBC.pdf"><h4>Restriction Digest</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/1/1e/Liquid_Liquid_Extraction_Protocol_UBC.pdf"><h4>Liquid-Liquid Extraction</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/b/b8/Preparation_of_Samples_to_run_SDS_UBC.pdf"><h4>Sample Prep for SDS</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/8/8e/Resting_Cell_Assay_UBC.pdf"><h4>Resting Cell Assay</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/2/2c/SDS_Gel_Prep_UBC.pdf"><h4>SDS Gel Prep</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/d/d7/Cell_Lysate_Experiment_of_CCH2_UBC.pdf"><h4>Cell Lysate Experiment</h4></a>
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<a href="#"><div class="hexagon"> <a href="https://static.igem.org/mediawiki/2015/7/7f/Determination_of_CCH2_Rate_UBC.pdf"><h4>CCH2 Rate Experiment</h4></a>
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Latest revision as of 22:26, 18 September 2015

UBC iGEM 2015

 

Protocols