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[DESCRIPTION]
<a href="https://www.neb.com/products/m0302-t7-endonuclease-i" target="_blank">T7 Endonuclease I</a>&nbsp;recognizes and cleaves non-perfectly matched DNA. This protocol describes how to determine genome targeting efficiency by digesting annealed PCR products with T7 Endonuclease I. In the first step PCR products are produced from the genomic DNA of cells whose genomes were targeted using Cas9, TALEN, ZFN etc. In the second step, the PCR products are annealed and digested with T7 Endonuclease I. Fragments are analyzed to determine the efficiency of genome targeting.

[GUIDELINES]
<strong>Overview:</strong><br /><br /><a href="https://www.neb.com/products/m0292t7endonucleasei" target="_blank">T7 Endonuclease I</a> recognizes and cleaves non-perfectly matched DNA. This protocol describes how to determine genome targeting efficiency by digesting annealed PCR products with T7 Endonuclease I. In the first step PCR products are produced from the genomic DNA of cells whose genomes were targeted using Cas9, TALEN, ZFN etc. In the second step, the PCR products are annealed and digested with T7 Endonuclease I. Fragments are analyzed to determine the efficiency of genome targeting.<br /><br /><strong>Required Materials:</strong><br />
<ul style="list-style-type: disc;">
<li><a href="https://www.neb.com/products/m0494-q5-hot-start-high-fidelity-2x-master-mix" target="_blank">Q5® Hot Start High-Fidelity 2X Master Mix (M0494S)</a></li>
<li>T7 Endonuclease I (M0302S)</li>
<li>0.25 mM EDTA</li>
<li>Purified genomic DNA from targeted cells</li>
<li>PCR primers to amplify a ~1kb region containing the target site</li>
<li>The target site should be offset from the center of the amplicon so that digestion produces easily resolvable DNA fragments</li>
<li>PCR primer design is critical. Please visit NEB's <a href="https://www.neb.com/tools-and-resources" target="_blank">Tools and Resources</a> page to optimize your primer design using the <a href="https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator" target="_blank">NEB Tm Calculator</a></li>
<li>A PCR thermocycler with programmable temperature ramp rate</li>
<li>DNA purification system - we recommend Ampure XP beads</li>
<li>Apparatus to quantitate DNA - spectrophotometer or fluorometer</li>
<li>Apparatus to analyze DNA fragments – e.g. Agilent Bioanalyzer, Qiagen Qiaxel, or standard agarose gel electrophoresis</li>
</ul>
<br /><strong><strong>PCR<br /><br /></strong>Cycling Conditions</strong>
<table>
<tbody>
<tr class="border">
<td><strong>Step</strong></td>
<td><strong>Temperature</strong></td>
<td><strong>Time</strong></td>
</tr>
<tr class="border">
<td>Initial Denaturation</td>
<td>98°C</td>
<td>30 seconds</td>
</tr>
<tr class="border">
<td rowspan="3">35 cycles<br /><br /></td>
<td>98°C</td>
<td>5 seconds</td>
</tr>
<tr class="border">
<td>*50-72°C</td>
<td>10 seconds</td>
</tr>
<tr class="border">
<td>72°C</td>
<td>20 seconds</td>
</tr>
<tr class="border">
<td>Final Extension</td>
<td>72°C</td>
<td>2 minutes</td>
</tr>
<tr class="border">
<td>Hold</td>
<td>4-10°C</td>
<td> </td>
</tr>
</tbody>
</table>
<strong><strong>*Use of the <a href="https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator" target="_blank">NEB Tm Calculator</a> is highly recommended.<br />Note: Q5 Hot Start High-Fidelity 2X Master Mix does not require a separate activation step. Standard Q5 cycling conditions are recommended.<br /><br />T7 Endonuclease I digestion:<br /><br />Anneal the PCR products in a thermocycler using the following conditions:<br /></strong>Hybridization Conditions</strong>
<table>
<tbody>
<tr class="border">
<td><strong>Step</strong></td>
<td><strong>Temperature</strong></td>
<td><strong>Ramp Rate</strong></td>
<td><strong>Time</strong></td>
</tr>
<tr class="border">
<td>Initial Denaturation</td>
<td>95°C</td>
<td> </td>
<td>5 minutes</td>
</tr>
<tr class="border">
<td rowspan="2">Annealing </td>
<td>95-85°C</td>
<td>-2°C/second</td>
<td> </td>
</tr>
<tr class="border">
<td>85-25°C</td>
<td>-0.1°C/second</td>
<td> </td>
</tr>
<tr class="border">
<td>Hold</td>
<td>4°C</td>
<td> </td>
<td>Hold</td>
</tr>
</tbody>
</table>
<strong><br class="Apple-interchange-newline" />REFERENCES:<br /></strong>Guschin, D.Y., et. al.(2010) <a href="http://link.springer.com/protocol/10.1007%2F978-1-60761-753-2_15" target="_blank">A rapid and general assay for monitoring endogenous gene modification</a>. Methods Mol Biol, 649, 247–256.<strong><br /></strong>

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