TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Adapted from Dignam JD, Lebovitz RM, Roeder RG. 1983.  <a href="https://www-ncbi-nlm-nih-gov.insb.bib.cnrs.fr/pubmed/6828386" target="_blank">Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.</a> Nucleic Acids Res. 11(5):1475-89. PMID:6828386</p>

[BEFORE_START]
<p> </p>
<p> </p>
<p>Prepare the following buffers and store at +4°C:</p>
<p> </p>
<table style="width: 473.734375px;">
<tbody>
<tr>
<td style="width: 466.734375px;" colspan="4">
<p><strong>PMSF 0.2 M:</strong></p>
<p>3.48 g /100 ml DMSO</p>
</td>
</tr>
<tr>
<td style="width: 170px;">
<p><strong>TGME:</strong></p>
<p>Glycerol</p>
<p>Tris, pH 7.9</p>
<p>MgCl<sub>2</sub></p>
<p>EDTA</p>
<p>water</p>
</td>
<td style="width: 62px; text-align: right;">
<p><strong>Stock</strong></p>
<p>100 %</p>
<p>0.5 M</p>
<p>1 M</p>
<p>0.5 M</p>
<p> </p>
</td>
<td style="width: 94px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>50 %</p>
<p>50 mM</p>
<p>5 mM</p>
<p>0.1 mM</p>
<p> </p>
</td>
<td style="width: 140.734375px; text-align: right;">
<p><strong>Vol (for 1 l)</strong></p>
<p>500 ml</p>
<p>100 ml</p>
<p>5 ml</p>
<p>0.2 ml</p>
<p>to 1 l</p>
</td>
</tr>
<tr>
<td style="width: 170px;">
<p><strong>Low Salt Buffer:</strong></p>
<p>Tris, pH 7.3</p>
<p>Glycerol</p>
<p>MgCl<sub>2</sub></p>
<p>EDTA pH 8</p>
<p>water</p>
</td>
<td style="width: 62px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>50 %</p>
<p>1 M</p>
<p>0.5 M</p>
<p> </p>
</td>
<td style="width: 94px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>20 mM</p>
<p>12.5 %</p>
<p>1.5 mM</p>
<p>0.2 mM</p>
<p> </p>
</td>
<td style="width: 140.734375px; text-align: right;">
<p><strong>Vol (for 1 l)</strong></p>
<p>20 ml</p>
<p>250 ml</p>
<p>1.5 ml</p>
<p>0.4 ml</p>
<p>to 1 l</p>
</td>
</tr>
<tr>
<td style="width: 170px;">
<p><strong>High Salt Buffer:</strong></p>
<p>Tris, pH 7.3</p>
<p>Glycerol</p>
<p>MgCl<sub>2</sub></p>
<p>EDTA pH 8</p>
<p>KCl</p>
<p>water</p>
</td>
<td style="width: 62px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>50 %</p>
<p>1 M</p>
<p>0.5 M</p>
<p>3 M</p>
<p> </p>
</td>
<td style="width: 94px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>20 mM</p>
<p>12.5 %</p>
<p>1.5 mM</p>
<p>0.2 mM</p>
<p>1.2 M</p>
<p> </p>
</td>
<td style="width: 140.734375px; text-align: right;">
<p><strong>Vol (for 1 l)</strong></p>
<p>20 ml</p>
<p>250 ml</p>
<p>1.5 ml</p>
<p>0.4 ml</p>
<p>400 ml</p>
<p>to 1 l</p>
</td>
</tr>
<tr>
<td style="width: 170px;">
<p><strong>Hypotonic Buffer (HB):</strong></p>
<p>Tris, pH 7.3</p>
<p>KCl</p>
<p>MgCl<sub>2</sub></p>
<p>water</p>
</td>
<td style="width: 62px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>3 M</p>
<p>1 M</p>
<p> </p>
</td>
<td style="width: 94px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>10 mM</p>
<p>10 mM</p>
<p>1.5 mM</p>
<p> </p>
</td>
<td style="width: 140.734375px; text-align: right;">
<p><strong>Vol (for 1 l)</strong></p>
<p>10 ml</p>
<p>3.34 ml</p>
<p>1.5 ml</p>
<p>to 1 l</p>
</td>
</tr>
<tr>
<td style="width: 170px;">
<p><strong>10 x Buffer </strong></p>
<p>Tris, pH 7.3</p>
<p>KCl</p>
<p>MgCl<sub>2</sub></p>
<p>water</p>
</td>
<td style="width: 62px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>3 M</p>
<p>1 M</p>
<p> </p>
</td>
<td style="width: 94px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>30 mM</p>
<p>140 mM</p>
<p>3 mM</p>
<p> </p>
</td>
<td style="width: 140.734375px; text-align: right;">
<p><strong>Vol (for 1 l)</strong></p>
<p>30 ml</p>
<p>46.6 ml</p>
<p>3 ml</p>
<p>to 1 l</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Cold room set up:</p>
<p>thaw 0.2 M PMSF</p>
<p>prepare hypotonic buffer (500 ml HB + 0.35 ml beta-ME + 0.5 ml 0.2 M PMSF)</p>
<p>prepare freezing solution (20 ml TGME + 0.4 ml DTT, in a 50 ml tube)</p>
<p>prepare tubes</p>

[GUIDELINES]
<p>The protocol is suitable for HeLa S3 cells. Parameters should be adapted for other cell types.</p>

[STEPS]
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