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 His10-MBP-Cas9 purification. </p>

[BEFORE_START]
<p><strong>DAY 1 materials</strong></p>
<p> </p>
<table style="height: 186px; width: 332px;">
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<td style="width: 180px;">
<div title="Page 1">
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<p><strong>Lysis Buffer</strong></p>
</div>
</div>
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</td>
<td style="width: 140px;"><strong>200 ml </strong></td>
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<td style="width: 180px;">50 mM Tris-Cl, pH 7.5</td>
<td style="width: 140px;">10 ml 4M</td>
</tr>
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<td style="width: 180px;">500 mM NaCl</td>
<td style="width: 140px;">25 ml 4M</td>
</tr>
<tr>
<td style="width: 180px;">1 mM TCEP</td>
<td style="width: 140px;">400 µl 0.5M </td>
</tr>
<tr>
<td style="width: 180px;">5% glycerol</td>
<td style="width: 140px;">10 ml 100%</td>
</tr>
<tr>
<td style="width: 180px;">0.5mM PMSF</td>
<td style="width: 140px;">250 µl 400 mM </td>
</tr>
<tr>
<td style="width: 180px;">protease inhibitors (Roche)</td>
<td style="width: 140px;"> 4 tablets</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 164px; width: 333px;">
<tbody>
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<td style="width: 181px;"><strong>Wash Buffer</strong></td>
<td style="width: 140px;"><strong>500 ml </strong></td>
</tr>
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<td style="width: 181px;">50 mM Tris-Cl, pH 7.5</td>
<td style="width: 140px;">25 ml 1M</td>
</tr>
<tr>
<td style="width: 181px;">500 mM NaCl</td>
<td style="width: 140px;">62.5 ml 4M</td>
</tr>
<tr>
<td style="width: 181px;">1 mM TCEP</td>
<td style="width: 140px;">1 ml </td>
</tr>
<tr>
<td style="width: 181px;">10 mM imidazole</td>
<td style="width: 140px;">1.67 ml 3M</td>
</tr>
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<td style="width: 181px;">5% glycerol</td>
<td style="width: 140px;">25 ml 100% </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 138px; width: 332px;">
<tbody>
<tr>
<td style="width: 184px;"><strong>Elution Buffer</strong></td>
<td style="width: 136px;"><strong>200 ml </strong></td>
</tr>
<tr>
<td style="width: 184px;">50 mM Tris-Cl, pH 7.5</td>
<td style="width: 136px;">10 ml 1M</td>
</tr>
<tr>
<td style="width: 184px;">500 mM NaCl</td>
<td style="width: 136px;">25 ml 4M</td>
</tr>
<tr>
<td style="width: 184px;">1 mM TCEP</td>
<td style="width: 136px;">0.4 ml 0.5M</td>
</tr>
<tr>
<td style="width: 184px;">300 mM imidazole</td>
<td style="width: 136px;">20 ml 3M</td>
</tr>
<tr>
<td style="width: 184px;">5% glycerol</td>
<td style="width: 136px;">10 ml 100%</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>DAY 2 materials</strong></p>
<p> </p>
<table style="height: 131px; width: 331px;">
<tbody>
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<td style="width: 185px;"><strong>Cas9 IEX Buffer A</strong></td>
<td style="width: 140px;"><strong>1500 ml</strong></td>
</tr>
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<td style="width: 185px;">20 mM Tris-Cl, pH 7.5</td>
<td style="width: 140px;">30 ml 1M</td>
</tr>
<tr>
<td style="width: 185px;">125 mM KCl</td>
<td style="width: 140px;">93.75 ml 2M</td>
</tr>
<tr>
<td style="width: 185px;">1 mM TCEP</td>
<td style="width: 140px;">3 ml 0.5M</td>
</tr>
<tr>
<td style="width: 185px;">5% glycerol</td>
<td style="width: 140px;">75ml 100%</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 120px; width: 330px;">
<tbody>
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<td style="width: 183px;"><strong>Cas9 IEX Buffer B</strong></td>
<td style="width: 143px;"><strong>500 ml</strong></td>
</tr>
<tr>
<td style="width: 183px;">20 mM Tris-Cl, pH 7.5</td>
<td style="width: 143px;">20 ml 1M</td>
</tr>
<tr>
<td style="width: 183px;">1 M KCl</td>
<td style="width: 143px;">250 ml 2M</td>
</tr>
<tr>
<td style="width: 183px;">1 mM TCEP</td>
<td style="width: 143px;">1 ml 0.5M</td>
</tr>
<tr>
<td style="width: 183px;">5% glycerol</td>
<td style="width: 143px;">25 ml 100%</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 124px; width: 329px;">
<tbody>
<tr>
<td style="width: 178px;"><strong>Cas9 Gel Filtration Buffer</strong></td>
<td style="width: 145px;"><strong>1000 ml </strong></td>
</tr>
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<td style="width: 178px;">20 mM Tris-Cl, pH 7.5</td>
<td style="width: 145px;">20 ml 1M</td>
</tr>
<tr>
<td style="width: 178px;">200 mM KCl</td>
<td style="width: 145px;">100 ml 2M</td>
</tr>
<tr>
<td style="width: 178px;">1 mM TCEP</td>
<td style="width: 145px;">2 ml 0.5M</td>
</tr>
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<td style="width: 178px;">5% glycerol</td>
<td style="width: 145px;">50 ml 100%</td>
</tr>
</tbody>
</table>

[GUIDELINES]
<p>This protocol takes two days to execute. </p>

[STEPS]
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