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[GUIDELINES]
<p style="text-align: left;">Modified from Marcia Osburne<br /><br />F1 = 5&rsquo;-CTTCCTCGTGAAGGTGGAA-3&rsquo; (&ldquo;published&rdquo;)</p>
<p>R1 = 5&rsquo;-CAGCGAAGTAATTGTCTGCAATATAATC-3&rsquo; (&ldquo;published&rdquo;)</p>
<p>F2 = 5&rsquo;-GTTGCCTAGAAGAGAAGGTGGTCG-3&rsquo; (&ldquo;genomic&rdquo;)</p>
<p>R2 = 5&rsquo;-CGGTGAAGTAGTTATCAGCAATGAAGTC-3&rsquo; (&ldquo;genomic&rdquo;)</p>
<p>Amplicons F1/R1 = 356bp and F2/R2 = 354bp<br /><br /><br /><strong>Marcia Osburne protocol (from 1/12/10 email):</strong></p>
<table>
<tbody>
<tr>
<td>Amt.</td>
<td>Reagent</td>
<td>Final Conc.</td>
<td>Cycling</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2&mu;l</td>
<td>template (i.e., lysate)</td>
<td>&nbsp;</td>
<td>94<sup>o</sup>C, 5min, then add Taq</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>5&mu;l</td>
<td>10 x Taq Buffer</td>
<td>1x</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2.5&mu;l</td>
<td>10mM dNTP</td>
<td>0.5mM</td>
<td>94<sup>o</sup>C, 1 min</td>
<td rowspan="3">35x</td>
</tr>
<tr>
<td>1&mu;l</td>
<td>100&mu;M 5' primer</td>
<td>2&mu;M</td>
<td>52<sup>o</sup>C, 1.5 min</td>
</tr>
<tr>
<td>1&mu;l</td>
<td>100&mu;M 3' primer</td>
<td>2&mu;M</td>
<td>72<sup>o</sup>C, 1.5 min</td>
</tr>
<tr>
<td>2.5&mu;l</td>
<td>50mM MgCl<sub>2</sub>&nbsp;</td>
<td>2.5mM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>35.5&mu;l</td>
<td>H<sub>2</sub>O</td>
<td>50&mu;l reaction volume</td>
<td>72<sup>o</sup>C, 10 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>0.5&mu;l</td>
<td>Taq (add after hot start)</td>
<td>2.5U/reaction</td>
<td>4<sup>o</sup>C, hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br /><strong>Takara LA HS Taq protocol:</strong><br />
<table>
<tbody>
<tr>
<td>Amt.</td>
<td>Reagent</td>
<td>Final Conc.</td>
<td>Cycling</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2&mu;l</td>
<td>template (i.e., lysate)</td>
<td>&nbsp;</td>
<td>94<sup>o</sup>C, 5min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>5&mu;l</td>
<td>10 x Taq HS LA Buffer</td>
<td>1x</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2.5&mu;l</td>
<td>10mM ea. dNTP</td>
<td>0.5mM ea.</td>
<td>94<sup>o</sup>C, 30 sec</td>
<td rowspan="3">35x</td>
</tr>
<tr>
<td>1&mu;l</td>
<td>100&mu;M 5' primer</td>
<td>2&mu;M</td>
<td>52<sup>o</sup>C, 1.5 min</td>
</tr>
<tr>
<td>1&mu;l</td>
<td>100&mu;M 3' primer</td>
<td>2&mu;M</td>
<td>72<sup>o</sup>C, 1.5 min</td>
</tr>
<tr>
<td>N/A</td>
<td>MgCl<sub>2</sub>&nbsp;</td>
<td>2.5mM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>35.5&mu;l</td>
<td>H<sub>2</sub>O</td>
<td>50&mu;l reaction volume</td>
<td>72<sup>o</sup>C, 10 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>0.5&mu;l</td>
<td>Takara LA Taq (5U/&mu;l)</td>
<td>2.5U/reaction</td>
<td>15<sup>o</sup>C, hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br /><strong>Sigma Taq protocol:</strong><br />
<table>
<tbody>
<tr>
<td>Amt.</td>
<td>Reagent</td>
<td>Final Conc.</td>
<td>Cycling</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1&mu;l</td>
<td>template (i.e., lysate)</td>
<td>&nbsp;</td>
<td>94<sup>o</sup>C, 5min, then add taq</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2.5&mu;l</td>
<td>10 x Buffer w/o MgCl<sub>2</sub></td>
<td>1x</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.25&mu;l</td>
<td>10mM ea. dNTP</td>
<td>0.5mM ea.</td>
<td>94<sup>o</sup>C, 30 sec</td>
<td rowspan="3">35x</td>
</tr>
<tr>
<td>0.5&mu;l</td>
<td>100&mu;M 5' primer</td>
<td>2&mu;M</td>
<td>52<sup>o</sup>C, 1.5 min</td>
</tr>
<tr>
<td>0.5&mu;l</td>
<td>100&mu;M 3' primer</td>
<td>2&mu;M</td>
<td>72<sup>o</sup>C, 1.5 min</td>
</tr>
<tr>
<td>2.5&mu;l</td>
<td>25mM MgCl<sub>2</sub>&nbsp;</td>
<td>2.5mM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>16.5&mu;l</td>
<td>H<sub>2</sub>O</td>
<td>25&mu;l reaction volume</td>
<td>72<sup>o</sup>C, 10 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>0.25&mu;l</td>
<td>Sigma Taq&nbsp;(5U/&micro;l)</td>
<td>2.5U/reaction</td>
<td>15<sup>o</sup>C, hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
Note: most "10mM dNTP" are 2.5mM per dNTP; try decreasing Sigma 10mM ea. dNTP (at. D7295) to 1&mu;l/50&mu;l reation which will be 0.2mM ea. in final reaction.&nbsp;<br /><strong>Conclusions from 1/18/2010 Exp. 108:</strong><br /><br />* &nbsp; Primers F1/R1 amplify JW Syn 26 lysate<br />* &nbsp; Primers F2/R2 amplify MBS Syn33a lysate<br />* &nbsp; JW Syn33 lysate not amplified by either primer set

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