TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol from the <a href="http://doudnalab.org/" target="_blank" rel="noopener noreferrer">Doudna Lab</a> for preparation of Cas9 protein. </p>

[BEFORE_START]
<div title="Page 1">
<p><strong>Materials</strong></p>
<p> </p>
<table style="height: 112px; width: 301px;">
<tbody>
<tr>
<td style="width: 144px;">
<div title="Page 1">
<div>
<div>
<p><strong>TY media</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 145px;"><strong>1 L</strong></td>
</tr>
<tr>
<td style="width: 144px;">
<div title="Page 1">
<div>
<div>
<p>Tryptone</p>
</div>
</div>
</div>
</td>
<td style="width: 145px;">10 g</td>
</tr>
<tr>
<td style="width: 144px;">
<div title="Page 1">
<div>
<div>
<p>Yeast extract</p>
</div>
</div>
</div>
</td>
<td style="width: 145px;">6 g</td>
</tr>
<tr>
<td style="width: 144px;">NaCl</td>
<td style="width: 145px;">10 g</td>
</tr>
<tr>
<td style="width: 289px;" colspan="2">
<div title="Page 1">
<div>
<div>
<p>Dissolve in 400 mL<br /> Rinse flasks thoroughly with MQ water</p>
<p>Add 600 mL MQ water, media</p>
<p>Autoclave (45’, no dry time)</p>
</div>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>Buffers </strong></p>
<p> </p>
<table style="height: 143px; width: 302px;">
<tbody>
<tr>
<td style="width: 146px;">
<div title="Page 1">
<div>
<div>
<p><strong>Lysis </strong></p>
</div>
</div>
</div>
</td>
<td style="width: 144px;"><strong>1L, pH7.5</strong></td>
</tr>
<tr>
<td style="width: 146px;">
<div title="Page 1">
<div>
<div>
<p>20mM HEPES</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">
<div title="Page 1">
<div>
<div>
<p>20 ml of 1M</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 146px;">
<div title="Page 1">
<div>
<div>
<p>1M KCl</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">74.55 g</td>
</tr>
<tr>
<td style="width: 146px;">
<div title="Page 1">
<div>
<div>
<p>10% glycerol</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">100 ml</td>
</tr>
<tr>
<td style="width: 146px;">
<div title="Page 1">
<div>
<div>
<p>5mM TCEP</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">1.43 g</td>
</tr>
<tr>
<td style="width: 146px;">
<div title="Page 1">
<div>
<div>
<p>10mM imidazole</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">680 mg </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 158px; width: 300px;">
<tbody>
<tr>
<td style="width: 147px;">
<div title="Page 1">
<div>
<div>
<p><strong>Elution</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 143px;"><strong>500mL, pH7.5</strong></td>
</tr>
<tr>
<td style="width: 147px;">
<div title="Page 1">
<div>
<div>
<p>20mM HEPES</p>
</div>
</div>
</div>
</td>
<td style="width: 143px;">10 ml of 1M</td>
</tr>
<tr>
<td style="width: 147px;">
<div title="Page 1">
<div>
<div>
<p>0.1M KCl</p>
</div>
</div>
</div>
</td>
<td style="width: 143px;">50 ml of 1M</td>
</tr>
<tr>
<td style="width: 147px;">
<div title="Page 1">
<div>
<div>
<p>10% glycerol</p>
</div>
</div>
</div>
</td>
<td style="width: 143px;">50 ml</td>
</tr>
<tr>
<td style="width: 147px;">
<div title="Page 1">
<div>
<div>
<p>5mM TCEP</p>
</div>
</div>
</div>
</td>
<td style="width: 143px;">717 mg</td>
</tr>
<tr>
<td style="width: 147px;">
<div title="Page 1">
<div>
<div>
<p>300mM imidazole</p>
</div>
</div>
</div>
</td>
<td style="width: 143px;">10.2 g </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 137px; width: 302px;">
<tbody>
<tr>
<td style="width: 148px;">
<div title="Page 1">
<div>
<div>
<p><strong>Ion Exchange buffer A</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">
<div title="Page 1">
<div>
<div>
<p><strong>1L, pH7.5 (low salt)</strong></p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 148px;">
<div title="Page 1">
<div>
<div>
<p>20mM HEPES</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">20 ml of 1M</td>
</tr>
<tr>
<td style="width: 148px;">
<div title="Page 1">
<div>
<div>
<p>300mM KCl</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">300 ml of 1M</td>
</tr>
<tr>
<td style="width: 148px;">
<div title="Page 1">
<div>
<div>
<p>10% glycerol</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">100 ml </td>
</tr>
<tr>
<td style="width: 148px;">
<div title="Page 1">
<div>
<div>
<p>1mM TCEP</p>
</div>
</div>
</div>
</td>
<td style="width: 144px;">287 mg</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 133px; width: 299px;">
<tbody>
<tr>
<td style="width: 150px;">
<div title="Page 1">
<div>
<div>
<p><strong>Ion Exchange buffer B</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 141px;">
<div title="Page 1">
<div>
<div>
<p><strong>1L, pH7.5 (high salt)</strong></p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 150px;">
<div title="Page 1">
<div>
<div>
<p>20mM HEPES</p>
</div>
</div>
</div>
</td>
<td style="width: 141px;">20 ml of 1M</td>
</tr>
<tr>
<td style="width: 150px;">
<div title="Page 1">
<div>
<div>
<p>1M KCl</p>
</div>
</div>
</div>
</td>
<td style="width: 141px;">74.55 g</td>
</tr>
<tr>
<td style="width: 150px;">
<div title="Page 1">
<div>
<div>
<p>10% glycerol</p>
</div>
</div>
</div>
</td>
<td style="width: 141px;">100 ml </td>
</tr>
<tr>
<td style="width: 150px;">
<div title="Page 1">
<div>
<div>
<p>1mM TCEP</p>
</div>
</div>
</div>
</td>
<td style="width: 141px;">287 ml</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 111px; width: 303px;">
<tbody>
<tr>
<td style="width: 139px;">
<div title="Page 1">
<div>
<div>
<p><strong>Gel Filtration</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 152px;">
<div title="Page 1">
<div>
<div>
<p><strong>1L, pH7.5</strong></p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 139px;">
<div title="Page 1">
<div>
<div>
<p>20mM HEPES</p>
</div>
</div>
</div>
</td>
<td style="width: 152px;">20 ml of 1M</td>
</tr>
<tr>
<td style="width: 139px;">
<div title="Page 1">
<div>
<div>
<p>150mM KCl</p>
</div>
</div>
</div>
</td>
<td style="width: 152px;">150 ml of 1M</td>
</tr>
<tr>
<td style="width: 139px;">
<div title="Page 1">
<div>
<div>
<p>10% glycerol</p>
</div>
</div>
</div>
</td>
<td style="width: 152px;">100 ml</td>
</tr>
<tr>
<td style="width: 139px;">
<div title="Page 1">
<div>
<div>
<p>1mM TCEP</p>
</div>
</div>
</div>
</td>
<td style="width: 152px;">287 mg</td>
</tr>
</tbody>
</table>
<p> </p>
</div>

[GUIDELINES]
<p>The protocol workflow is as follows: </p>
<p> </p>
<p><strong>1. Expression of Cas9 in Rosetta2 E. coli</strong></p>
<ul>
<li>Transformation (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=1" target="_blank" rel="noopener noreferrer">Steps 1-5</a>)</li>
<li>Starter culture (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=6" target="_blank" rel="noopener noreferrer">Steps 6-8</a>)</li>
<li title="Page 2">
<p>Expression (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=9" target="_blank" rel="noopener noreferrer">Steps 9-21</a>)</p>
</li>
</ul>
<p> </p>
<p><strong>2. Purification of Cas9</strong> </p>
<ul>
<li>Sonication (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=22" target="_blank" rel="noopener noreferrer">Steps 22-29</a>)</li>
<li>HisTrap (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=30" target="_blank" rel="noopener noreferrer">Steps 30-40</a>)</li>
<li>TEV (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=41" target="_blank" rel="noopener noreferrer">Steps 41-45</a>)</li>
<li>Heparin (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=46" target="_blank" rel="noopener noreferrer">Steps 46-53</a>)</li>
<li title="Page 3">
<p>BPTrap/Superdex 200 (<a href="https://www.protocols.io/view/preparation-of-cas9-protein-rczd2x6?step=54" target="_blank" rel="noopener noreferrer">Steps 54-65</a>)</p>
<div title="Page 3"> </div>
</li>
</ul>

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