TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for <em>in vitro</em> transcription of DNA oligos by T7 polymerase from the <a href="http://doudnalab.org/" target="_blank" rel="noopener noreferrer">Doudna Lab</a>. </p>

[BEFORE_START]
<p><strong>Stock solutions: </strong></p>
<p> </p>
<table style="height: 164px; width: 300px;">
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<td style="width: 198px;">
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<p><strong>5x transcription buffer:</strong></p>
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</div>
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</td>
<td style="width: 98px;">
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<p>10 ml</p>
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</div>
</div>
</td>
</tr>
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<td style="width: 198px;">
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<p>150 mM Tris-Cl, pH 8.1</p>
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</div>
</div>
</td>
<td style="width: 98px;">
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<p>1.5 ml</p>
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</div>
</div>
</td>
</tr>
<tr>
<td style="width: 198px;">
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<div>
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<p>125 mM MgCl<sub>2</sub></p>
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</div>
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</td>
<td style="width: 98px;">
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<p>1.25 ml</p>
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</div>
</div>
</td>
</tr>
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<td style="width: 198px;">
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<p>0.05% Triton X-100</p>
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</div>
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</td>
<td style="width: 98px;">
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<p>50 µl</p>
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</div>
</div>
</td>
</tr>
<tr>
<td style="width: 198px;">
<div title="Page 1">
<div>
<div>
<p>10 mM spermidine</p>
</div>
</div>
</div>
</td>
<td style="width: 98px;">
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<div>
<p>100 µl</p>
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</div>
</div>
</div>
</td>
</tr>
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<td style="width: 198px;">
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<div>
<div>
<p>H<sub>2</sub>O</p>
</div>
</div>
</div>
</td>
<td style="width: 98px;">to 10mL</td>
</tr>
<tr>
<td style="width: 198px;">
<div title="Page 1">
<div>
<div>
<p>store at -20 °C</p>
</div>
</div>
</div>
</td>
<td style="width: 98px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 131px; width: 300px;">
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<td style="width: 196px;">
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<p><strong>10x TBE </strong></p>
</div>
</div>
</div>
</td>
<td style="width: 98px;">1 L</td>
</tr>
<tr>
<td style="width: 196px;">
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<p>Tris base</p>
</div>
</div>
</div>
</td>
<td style="width: 98px;">108 g</td>
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<td style="width: 196px;">
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<p>Boric acid</p>
</div>
</div>
</div>
</td>
<td style="width: 98px;">55 g</td>
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<td style="width: 196px;">
<div title="Page 1">
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<p>0.5 M Na<sub>2</sub>EDTA pH 8.0</p>
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</div>
</td>
<td style="width: 98px;">40 ml</td>
</tr>
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<td style="width: 196px;">
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<p>H<sub>2</sub>O</p>
</div>
</div>
</div>
</td>
<td style="width: 98px;">to 1 L</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 90px; width: 407px;">
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<tr>
<td style="width: 319px;">
<div title="Page 1">
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<div>
<p><strong>0.5 M Na<sub>2</sub>EDTA </strong></p>
</div>
</div>
</div>
</td>
<td style="width: 102px;">0.5 L</td>
</tr>
<tr>
<td style="width: 319px;">
<div title="Page 1">
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<div>
<p>Disodium ethylenediaminetetraacetate 2H<sub>2</sub>O</p>
</div>
</div>
</div>
</td>
<td style="width: 102px;">93 g</td>
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<tr>
<td style="width: 319px;">
<div title="Page 1">
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<div>
<p>NaOH to get pH 8.0</p>
</div>
</div>
</div>
</td>
<td style="width: 102px;">10 g</td>
</tr>
<tr>
<td style="width: 319px;">H<sub>2</sub>O</td>
<td style="width: 102px;">to 500 ml </td>
</tr>
<tr>
<td style="width: 319px;">autoclave</td>
<td style="width: 102px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 84px; width: 252px;">
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<tr>
<td style="width: 171px;">
<div title="Page 2">
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<p><strong>10 % (w/v) APS</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 79px;">50 ml </td>
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<tr>
<td style="width: 171px;">
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<div>
<p>Ammonium persulfate</p>
</div>
</div>
</div>
</td>
<td style="width: 79px;">5 g</td>
</tr>
<tr>
<td style="width: 171px;">H<sub>2</sub>O</td>
<td style="width: 79px;">to 50 ml </td>
</tr>
<tr>
<td style="width: 171px;">store at -20 °C</td>
<td style="width: 79px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 104px; width: 215px;">
<tbody>
<tr>
<td style="width: 135px;">
<div title="Page 2">
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<p><strong>1 M spermidine</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 68px;"> </td>
</tr>
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<td style="width: 135px;">
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<p>Spermidine</p>
</div>
</div>
</div>
</td>
<td style="width: 68px;">0.7 g</td>
</tr>
<tr>
<td style="width: 135px;">H<sub>2</sub>O</td>
<td style="width: 68px;">to 5 ml </td>
</tr>
<tr>
<td style="width: 135px;">store at -20 °C</td>
<td style="width: 68px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 85px; width: 192px;">
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<td style="width: 98px;">
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<p><strong>5N NaOH</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 84px;">25 ml </td>
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<td style="width: 98px;">
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<p>NaOH</p>
</div>
</div>
</div>
</td>
<td style="width: 84px;">5 g</td>
</tr>
<tr>
<td style="width: 98px;">H<sub>2</sub>O</td>
<td style="width: 84px;"> to 25 ml </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 80px; width: 181px;">
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<td style="width: 93px;"><strong>1N NaOH</strong></td>
<td style="width: 80px;">20 ml</td>
</tr>
<tr>
<td style="width: 93px;">
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<div>
<p>5N NaOH</p>
</div>
</div>
</div>
</td>
<td style="width: 80px;">5 ml </td>
</tr>
<tr>
<td style="width: 93px;">H<sub>2</sub>O</td>
<td style="width: 80px;">to 20 ml</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 132px; width: 246px;">
<tbody>
<tr>
<td style="width: 165px;">
<div title="Page 2">
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<p><strong>10 % Triton X-100</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 75px;">10 ml</td>
</tr>
<tr>
<td style="width: 165px;">
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<p>Triton X-100</p>
</div>
</div>
</div>
</td>
<td style="width: 75px;">1 ml</td>
</tr>
<tr>
<td style="width: 165px;">H<sub>2</sub>O</td>
<td style="width: 75px;">9 ml</td>
</tr>
<tr>
<td style="width: 165px;">
<div title="Page 2">
<div>
<div>
<p>store at -20 °C</p>
</div>
</div>
</div>
</td>
<td style="width: 75px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 95px; width: 329px;">
<tbody>
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<td style="width: 232px;">
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<div>
<p><strong>1% (w/v) bromophenol blue</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 81px;">10 ml</td>
</tr>
<tr>
<td style="width: 232px;">
<div title="Page 2">
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<div>
<p>bromophenol blue</p>
</div>
</div>
</div>
</td>
<td style="width: 81px;">100 mg</td>
</tr>
<tr>
<td style="width: 232px;">H<sub>2</sub>O</td>
<td style="width: 81px;"> to 10 ml</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 84px; width: 316px;">
<tbody>
<tr>
<td style="width: 209px;">
<div title="Page 2">
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<div>
<p><strong>1% (w/v) xylene cyanol</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 95px;">10 ml</td>
</tr>
<tr>
<td style="width: 209px;">
<div title="Page 2">
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<div>
<p>xylene cyanol</p>
</div>
</div>
</div>
</td>
<td style="width: 95px;">100 mg</td>
</tr>
<tr>
<td style="width: 209px;">H<sub>2</sub>O</td>
<td style="width: 95px;"> to 10 ml</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<table style="height: 189px; width: 340px;">
<tbody>
<tr>
<td style="width: 213px;">
<div title="Page 2">
<div>
<div>
<p><strong>2x RNA Loading Dye</strong></p>
</div>
</div>
</div>
</td>
<td style="width: 113px;">30 ml </td>
</tr>
<tr>
<td style="width: 213px;">
<div title="Page 2">
<div>
<div>
<p>95% formamide</p>
</div>
</div>
</div>
</td>
<td style="width: 113px;">28.5 ml</td>
</tr>
<tr>
<td style="width: 213px;">
<div title="Page 2">
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<div>
<p>1 mM EDTA</p>
</div>
</div>
</div>
</td>
<td style="width: 113px;">
<div title="Page 2">
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<p>60 μl of 0.5 M</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 213px;">
<div title="Page 2">
<div>
<div>
<p>0.025% SDS</p>
</div>
</div>
</div>
</td>
<td style="width: 113px;">
<div title="Page 2">
<div>
<div>
<p>75 μl of 10%</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 213px;">
<div title="Page 2">
<div>
<div>
<p>0.025% bromophenol blue</p>
</div>
</div>
</div>
</td>
<td style="width: 113px;">
<div title="Page 2">
<div>
<div>
<p>750 μl of 1%</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 213px;">
<div title="Page 2">
<div>
<div>
<p>0.01% xylene cyanol</p>
</div>
</div>
</div>
</td>
<td style="width: 113px;">
<div title="Page 2">
<div>
<div>
<p>300 μl of 1%</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 213px;">
<div title="Page 2">
<div>
<div>
<p>store at -20 °C</p>
</div>
</div>
</div>
</td>
<td style="width: 113px;"> </td>
</tr>
</tbody>
</table>

[GUIDELINES]
<div title="Page 1">
<div>
<div>
<p><strong>Template design:</strong></p>
<p><br />T7 polymerase requires a double-stranded promoter region for efficient template binding, but can then transcribe a single-stranded DNA template. Therefore, the following protocol uses an approach, whereby two oligos are annealed in order to generate a dsDNA promoter, followed by a single-stranded overhang that contains the reverse complement of the desired RNA sequence.</p>
</div>
</div>
<div>
<ul>
<li>The reverse complement of the desired RNA sequence is ordered as a DNA oligo (assuming the length does not exceed ~80 nt), to which the reverse complement of the T7 promoter is appended at the 3’ end
<ul>
<li>Example template: </li>
</ul>
</li>
</ul>
<p>                      <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/2h4fw26.png" width="478" height="108" data-src="https://s3.amazonaws.com/pr-journal/2h3fw26.png" data-ofn="Screen Shot 2018-06-28 at 1.33.47 PM.png" /></p>
</div>
<div>
<div title="Page 1">
<ul>
<li>A separate, synthetic DNA oligo is ordered that contains the T7 promoter in the forward direction, which can be annealed with the above construct:
<ul>
<li>T7oligo: 5’-TAATACGACTCACTATA-3’</li>
<li><strong>Note</strong>: according to Ambion, for transcription of synthetic oligonucleotides, only the -17 to -1 positions of the promoter need to be double-stranded. This oligo takes this into account.</li>
</ul>
</li>
</ul>
</div>
</div>
</div>

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