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[DESCRIPTION]
The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.

[GUIDELINES]
<strong>Guidelines for Optimizing Restriction Endonuclease Reactions<br /></strong><br /> If you are using a Master Mix, see "<a href="https://www.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions-when-using-re-mix" target="_blank">optimizing RE-Mix&reg; reactions</a>". <br /><br />There are several key factors to consider when setting up a restriction endonuclease digest. Using the proper amounts of DNA, enzyme and buffer components in the correct reaction volume will allow you to achieve optimal digestion. By definition, 1 unit of restriction enzyme will completely digest 1 &mu;g of substrate DNA in a 50 &mu;l reaction in 60 minutes. This enzyme : DNA : reaction volume ratio can be used as a guide when designing reactions. However, most researchers follow the "typical" reaction conditions listed, where a 5&ndash;10 fold overdigestion is recommended to overcome variability in DNA source, quantity and purity. NEB offers the following tips to help you to achieve maximal success in your restriction endonuclease reactions. <br /><br /><strong>A "Typical" Restriction Digest</strong>
<table class="t1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td class="td1" valign="top">Restriction Enzyme</td>
<td class="td2" valign="top">10 units is sufficient, generally 1&micro;l is used</td>
</tr>
<tr>
<td class="td3" valign="top">DNA</td>
<td class="td4" valign="top">1 &micro;g</td>
</tr>
<tr>
<td class="td1" valign="top">10X NEBuffer</td>
<td class="td2" valign="top">5 &micro;l (1X)</td>
</tr>
<tr>
<td class="td3" valign="top">Total Reaction Volume</td>
<td class="td4" valign="top">50 &micro;l</td>
</tr>
<tr>
<td class="td1" valign="top">Incubation Time</td>
<td class="td2" valign="top">1 hour*</td>
</tr>
<tr>
<td class="td3" valign="top">Incubation Temperature</td>
<td class="td4" valign="top">Enzyme dependent</td>
</tr>
</tbody>
</table>
* Can be decreased to 5-15 minutes by using a <a href="https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/time-saver-qualified-restriction-enzymes/time-saver-qualified-restriction-enzymes" target="_blank">Time-Saver&trade; Qualified enzyme</a>. <br /><br /><strong>Enzyme</strong> <br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Keep on ice when not in the freezer&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Should be the last component added to reaction&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Follow with a quick ("touch") spin-down in a microcentrifuge.&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Do not vortex the reaction.</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">In general, we recommend 5&ndash;10 units of enzyme per &micro;g DNA, and 10&ndash;20 units for genomic DNA in a 1 hour digest.&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">NEB has introduced a line of </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/high-fidelity-restriction-enzymes/high-fidelity-restriction-endonucleases" target="_blank">High-Fidelity (HF&reg;) enzymes</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;"> that provide added flexibility to reaction setup.&nbsp;</span></li>
</ul>
<br /><strong>DNA</strong> <br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents or excessive salts. </span><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Extra wash steps during purification are recommended.&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Methylation of DNA can inhibit digestion with certain enzymes. For more information about methylation, </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/tools-and-resources/usage-guidelines/effects-of-cpg-methylation-on-restriction-enzyme-cleavage" target="_blank">Effect of CpG Methylation on Restriction Enzyme Cleavage</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;"> and </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/tools-and-resources/usage-guidelines/dam-and-dcm-methylases-of-e-coli" target="_blank">Dam and Dcm Methylases of <em>E.coli</em></a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">&nbsp;</span></li>
</ul>
<br /><strong>Buffer</strong> <br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Use at a 1X concentration&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Supplement with SAM (S-Adenosyl methionine) to the recommended concentration if required.&nbsp;</span></li>
</ul>
<br /><strong>Reaction Volume </strong><br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">A 50 &micro;l reaction volume is recommended for digestion of 1 &micro;g of substrate&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Enzyme volume should not exceed 10% of the total reaction volume to prevent </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/tools-and-resources/usage-guidelines/star-activity" target="_blank">star activity</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;"> due to excess glycerol&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Additives in the restriction enzyme storage buffer (e.g., glycerol, salt) as well as contaminants found in the substrate solution (e.g., salt, EDTA, or alcohol) can be problematic in smaller reaction volumes. The following guidelines can be used for techniques that require smaller reaction volumes.&nbsp;</span></li>
</ul>
<table class="t1" style="height: 116px;" width="427" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td class="td1" valign="top">&nbsp;</td>
<td class="td2" valign="top"><strong>Restriction Enzyme*</strong></td>
<td class="td3" valign="top"><strong>DNA</strong></td>
<td class="td4" valign="top"><strong>10X NEBuffer</strong></td>
</tr>
<tr>
<td class="td5" valign="top">10 &micro;l rxn**</td>
<td class="td6" valign="top">1 unit</td>
<td class="td7" valign="top">0.1 &micro;g</td>
<td class="td8" valign="top">1 &micro;l</td>
</tr>
<tr>
<td class="td9" valign="top">25 &micro;l rxn</td>
<td class="td10" valign="top">5 units</td>
<td class="td11" valign="top">0.5 &micro;g</td>
<td class="td12" valign="top">2.5 &micro;l</td>
</tr>
<tr>
<td class="td5" valign="top">50 &micro;l rxn</td>
<td class="td6" valign="top">10 units</td>
<td class="td7" valign="top">1 &micro;g</td>
<td class="td8" valign="top">5 &micro;l</td>
</tr>
</tbody>
</table>
* Restriction Enzymes can be diluted using the recommended diluent buffer when smaller amounts are needed.<br /> ** 10 &micro;l rxns should not be incubated for longer than 1 hour to avoid evaporation. <br /><br /><strong>Incubation</strong> <strong>Time</strong> <br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Incubation time is typically 1 hour&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Can often be decreased by using an excess of enzyme, or by using one of our </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/time-saver-qualified-restriction-enzymes" target="_blank">Time-Saver Qualified enzymes</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">.&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">It is possible, with many enzymes, to use fewer units and digest for up to 16 hours. For more information, visit </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/tools-and-resources/usage-guidelines/restriction-endonucleases-survival-in-a-reaction" target="_blank">Extended Digests with Restriction Endonucleases</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">.&nbsp;</span></li>
</ul>
<br /><strong>Stopping a Reaction<br /></strong><br /> If no further manipulation of DNA is required: <br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Terminate with a stop solution (10 &micro;l per 50 &micro;l rxn) [1x: 2.5% Ficoll&reg;-400, 10mM EDTA, 3.3mM Tris-Hcl, 0.08% SDS, 0.02% Dye 1, 0.001% Dye 2, pH 8.0@25&deg;C] (e.g., NEB </span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/products/b7024-gel-loading-dye-purple-6x" target="_blank">#B7024</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;"> )&nbsp;</span></li>
</ul>
<br />When further manipulation of DNA is required: <br /><br />
<ul style="list-style-type: circle;">
<li><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/tools-and-resources/usage-guidelines/heat-inactivation">Heat inactivation</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;"> can be used&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Remove enzyme by using a spin column or phenol/chloroform extraction&nbsp;</span></li>
</ul>
<br /><strong>Storage </strong><br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">S</span><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">torage at -20&deg;C is recommended for most restriction enzymes. For a few enzymes, storage at-70&deg;C is recommended for periods longer than 30 days. Please refer to the enzyme's technical data sheet or catalog entry for storage information.&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">10X NEBuffers should also be stored at -20&deg;C&nbsp;</span></li>
</ul>
<br /><strong>Stability</strong> <br /><br />All enzymes are assayed for activity every 4 months. The expiration date is found on the label. <br />Exposure to temperatures above -20&deg;C should be minimized whenever possible <br /><br /><strong>Control Reactions</strong><br /> <br />If you are having difficulty cleaving your DNA substrate, we recommend the following control reactions: <br /><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability&nbsp;</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.&nbsp;</span></li>
</ul>

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