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| − | <header class="group">
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| − | <li><a data-ga-category="Content Action" data-ga-action="Email" data-ga-label="/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273" href="https://www.neb.com/account/email-page?device=modal" class="email iframe modal h600 ga-event dialoged">Email</a></li><li><a data-ga-category="Content Action" data-ga-action="Add to My NEB" data-ga-label="/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273" class="favorite favorite" rel="{1E226F71-2246-436B-ABC7-0B5CA5805A12}" href="javascript:void(0)">My NEB</a></li><li><a data-ga-category="Content Action" data-ga-action="Print" data-ga-label="/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273" href="javascript:window.print()" class="print ga-event">Print</a></li><li><a data-ga-category="Content Action" data-ga-action="Download" data-ga-label="PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (M0273)" rel="/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273?device=pdf" class="pdf ga-event" href="javascript:void(0)">PDF</a></li>
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| − | </menu><h1>
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| − | PCR Protocol for <em>Taq</em> DNA Polymerase with Standard <em>Taq</em> Buffer (M0273)
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| − | </h1>
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| − | </header>
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| − | <p></p><h2>Overview</h2>
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| − | <p><strong>PCR </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> <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°C). <d>
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| − | </d>
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| − | </p><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 μl reaction</strong></td>
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| − | <td><strong>50 μl 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 μl </td>
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| − | <td valign="top">5 μl </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 µl</td>
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| − | <td>1 μl</td>
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| − | <td>200 µM</td>
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| − | </tr>
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| − | <tr>
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| − | <td>10 µM Forward Primer</td>
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| − | <td>0.5 µl</td>
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| − | <td>1 μl</td>
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| − | <td>0.2 µM (0.05–1 µM)</td>
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| − | </tr>
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| − | <tr>
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| − | <td>10 µM Reverse Primer</td>
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| − | <td>0.5 µl</td>
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| − | <td>1 μl</td>
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| − | <td>0.2 µM (0.05–1 µM)</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><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 µl</td>
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| − | <td>0.25 µl</td>
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| − | <td>1.25 units/50 µl 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 µl</td>
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| − | <td>to 50 µl</td>
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| − | <td> </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 the block preheated to 95°C and begin thermocycling. <br>
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| − | <br>
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| − | <strong>Thermocycling conditions for a routine PCR: </strong><br>
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| − | <br>
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| − | <table height="162" width="309">
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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°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°C<br>
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| − | 45-68°C<br>
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| − | 68°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°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°C </td>
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| − | <td> </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> <p></p>
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| − | <ol>
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| − | <li>
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| − | Template: <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 μl reaction are as follows:
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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–1 μg</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–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: <br>
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| − | <br>
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| − | Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (<a data-ga-label="http://frodo.wi.mit.edu/primer3" data-ga-action="https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273" data-ga-category="External Link" class="ga-event" 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 0.05–1 μM, typically 0.1–0.5 μM.<br>
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| − | <br>
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| − | </li>
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| − | <li>Mg<sup>++ </sup>and additives: <br>
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| − | <br>
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| − | Mg<sup>++ </sup>concentration of 1.5–2.0 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>. <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 μM 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: <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 μl reaction). However, the optimal concentration of <em>Taq</em> DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 μl reaction) in specialized applications.<br>
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| − | <br>
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| − | </li>
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| − | <li>Denaturation: <br>
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| − | <br>
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| − | An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended. <br>
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| − | <br>
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| − | During thermocycling a 15–30 second denaturation at 95°C is recommended.<br>
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| − | <br>
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| − | </li>
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| − | <li>Annealing: <br>
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| − | <br>
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| − | The annealing step is typically 15–60 seconds. Annealing temperature is based on the T<sub>m</sub> of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated T<sub>m</sub>. 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°C are used, a 2-step PCR protocol is possible (see #10). <br>
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| − | <br>
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| − | </li>
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| − | <li>Extension: <br>
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| − | <br>
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| − | The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.<br>
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| − | <br>
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| − | </li>
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| − | <li>Cycle number: <br>
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| − | <br>
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| − | Generally, 25–35 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: <br>
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| − | <br>
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| − | When primers with annealing temperatures above 65°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: </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°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°C<br>
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| − | 65-68°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°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°C <br>
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| − | </td>
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| − | <td> </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: <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´–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. 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. 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></p>
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| − |
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| − | </section>
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