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[DESCRIPTION]
Modified from H. Ochman protocol: <em>Environ. Micro. 2009 Kunin, Engelbrekson Ochman &amp; Hugenholt</em>z

[GUIDELINES]
<strong>Protocol Using Takara Hot Start EX Taq (cat. no RR006A)</strong><br /><br />
<table style="height: 250px; width: 250px;" width="416">
<tbody>
<tr>
<td colspan="2"><strong>Pyrotag PCR Master Mix</strong></td>
</tr>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Volume&nbsp;&mu;l</strong></td>
</tr>
<tr>
<td>Water</td>
<td>18.25</td>
</tr>
<tr>
<td>10x EX Buffer II</td>
<td>2.5</td>
</tr>
<tr>
<td>dNTP mix (2.5mM ea)</td>
<td>2</td>
</tr>
<tr>
<td>F primer (10&nbsp;&mu;M)</td>
<td>0.5</td>
</tr>
<tr>
<td>R primer (10&nbsp;&mu;M)</td>
<td>0.5</td>
</tr>
<tr>
<td>Taq (5U/&nbsp;&mu;l)</td>
<td>0.25</td>
</tr>
<tr>
<td>DNA template</td>
<td>1</td>
</tr>
</tbody>
</table>
<br />
<table style="width: 300px; height: 250px;">
<tbody>
<tr>
<td colspan="3"><strong>Cycling Conditions</strong></td>
</tr>
<tr>
<td><strong>Temp.&nbsp; &deg;C </strong></td>
<td><strong>Time (min)</strong></td>
<td><strong>Cycles</strong></td>
</tr>
<tr>
<td>95</td>
<td>3:00</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>95</td>
<td>:30</td>
<td rowspan="3">25<br /><br /><br /></td>
</tr>
<tr>
<td>55</td>
<td>:45</td>
</tr>
<tr>
<td>72</td>
<td>1:30</td>
</tr>
<tr>
<td>72</td>
<td>10:00</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>15</td>
<td>Hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br /><strong><br />Protocol Using Sigma Taq (cat. no. D4545) and dNTP mix (cat. no. D7295)</strong><br /><br />
<table style="width: 250px; height: 250px;">
<tbody>
<tr>
<td colspan="2"><strong>Pyrotag PCR Master Mix</strong></td>
</tr>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Volume &mu;l</strong></td>
</tr>
<tr>
<td>Water</td>
<td>17.75</td>
</tr>
<tr>
<td>10X Buffer w/o MgCl2</td>
<td>2.5</td>
</tr>
<tr>
<td>MgCl2 (25mM)</td>
<td>2</td>
</tr>
<tr>
<td>dNTP mix (10mM ea)</td>
<td>0.5</td>
</tr>
<tr>
<td>F primer (10&nbsp;&mu;M)</td>
<td>0.5</td>
</tr>
<tr>
<td>R primer (10 &mu;M)</td>
<td>0.5</td>
</tr>
<tr>
<td>Taq (5U/&mu;l)</td>
<td>0.25</td>
</tr>
<tr>
<td>DNA template</td>
<td>1</td>
</tr>
</tbody>
</table>
<table style="width: 300px; height: 250px;">
<tbody>
<tr>
<td colspan="3"><strong>Cycling Conditions</strong></td>
</tr>
<tr>
<td><strong>Temp.&nbsp;&nbsp;&deg;C&nbsp;&nbsp;</strong></td>
<td><strong>Time (min)</strong></td>
<td><strong>Cycles</strong></td>
</tr>
<tr>
<td>95</td>
<td>3:00</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>95</td>
<td>:30</td>
<td rowspan="3">25<br /><br /><br /></td>
</tr>
<tr>
<td>55</td>
<td>:45</td>
</tr>
<tr>
<td>72</td>
<td>1:30</td>
</tr>
<tr>
<td>72</td>
<td>10:00</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>15</td>
<td>Hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br /> Reconditioning PCR sample (from Nucleic Acids Research 2002 Thompson, Marcelino &amp; Polz):&nbsp;Make a 1:10 dilution of PCR product into fresh PCR mix; cycle at 95&deg;C for 3 minutes, then 3 cycles of 95&deg;C for 30 second, 55&deg;C for 45 seconds, and 72&deg;C for 1.30 minutes, followed by 10 minutes at 72&deg;C. &nbsp;<br /><br /><strong>Howard's Protocol and Primers for 16s Pyrotagged PCR:</strong><br /><br />
<table style="width: 250px; height: 250px;">
<tbody>
<tr>
<td colspan="2"><strong>Pyrotag PCR Master Mix</strong></td>
</tr>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Amount &mu;l</strong></td>
</tr>
<tr>
<td>Water</td>
<td>39.25</td>
</tr>
<tr>
<td>10x Buffer</td>
<td>5</td>
</tr>
<tr>
<td>dNTPs (10mM)</td>
<td>1</td>
</tr>
<tr>
<td>F primer (10 &mu;M)</td>
<td>0.5</td>
</tr>
<tr>
<td>R primer (10 &mu;M)</td>
<td>0.5</td>
</tr>
<tr>
<td>BSA (10mg/mL)</td>
<td>1.5</td>
</tr>
<tr>
<td>Taq (5u/UL</td>
<td>0.25</td>
</tr>
<tr>
<td>DNA</td>
<td>2</td>
</tr>
</tbody>
</table>
<br />
<table style="width: 300px; height: 300px;">
<tbody>
<tr>
<td colspan="3"><strong>Cycling Conditions</strong></td>
</tr>
<tr>
<td><strong>Temp.&nbsp;&nbsp;&deg;C&nbsp;&nbsp;</strong></td>
<td><strong>Time (min)</strong></td>
<td><strong>Cycles</strong></td>
</tr>
<tr>
<td>95</td>
<td>3:00</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>95</td>
<td>:30</td>
<td rowspan="3">25<br /><br /><br /></td>
</tr>
<tr>
<td>55</td>
<td>:45</td>
</tr>
<tr>
<td>72</td>
<td>1:30</td>
</tr>
<tr>
<td>72</td>
<td>10:00</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>4</td>
<td>Hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br /><strong>Pyrotag Primers: 5' - emulsion PCR primer/ unique tag/ universal primer - 3'</strong><br /><br /><strong>926 forward primers: </strong><br />926fA1 : gcc tcc ctc gcg cca tca g <span style="color: red;">agc</span> aaa ctY aaa Kga att gac gg &nbsp; <br />926fA2 : gcc tcc ctc gcg cca tca g <span style="color: red;">atg </span>aaa ctY aaa Kga att gac gg &nbsp; <br />926fA3 : gcc tcc ctc gcg cca tca g <span style="color: red;">ctc </span>aaa ctY aaa Kga att gac gg &nbsp; <br />926fA4 : gcc tcc ctc gcg cca tca g <span style="color: red;">cag</span> aaa ctY aaa Kga att gac gg &nbsp; <br />926fA5 : gcc tcc ctc gcg cca tca g <span style="color: red;">tac</span> aaa ctY aaa Kga att gac gg &nbsp; <br />926fA6 : gcc tcc ctc gcg cca tca g <span style="color: red;">tcg</span> aaa ctY aaa Kga att gac gg &nbsp; <br />926fA7 : gcc tcc ctc gcg cca tca g <span style="color: red;">act</span> aaa ctY aaa Kga att gac gg &nbsp;<br /> 926fA8 : gcc tcc ctc gcg cca tca g <span style="color: red;">cgt</span> aaa ctY aaa Kga att gac gg &nbsp;<br /> 926fA9 : gcc tcc ctc gcg cca tca g <span style="color: red;">acacg</span> aaa ctY aaa Kga att gac gg &nbsp; <br />926fA10 : gcc tcc ctc gcg cca tca g <span style="color: red;">tgctg</span> aaa ctY aaa Kga att gac gg &nbsp; <br />926fA11 : gcc tcc ctc gcg cca tca g <span style="color: red;">cagcaaa</span> aaa ctY aaa Kga att gac gg &nbsp;<br /> 926fA12 : gcc tcc ctc gcg cca tca g <span style="color: red;">catgaaa</span> aaa ctY aaa Kga att gac gg &nbsp; &nbsp; <br /><br /><strong>1492 reverse primer: </strong><br />1492rB1 : gcc ttg cca gcc cgc tca g <span style="color: red;">agc</span> tac ggY tac ctt gtt acg act t &nbsp; <br /><br /><br /><strong>Summary of PCR Conditions</strong><br /><br />
<table style="width: 486px; height: 412px;">
<tbody>
<tr>
<td>Reagent</td>
<td>Howard Ochman</td>
<td>EX Takara HS</td>
<td>Sigma Taq</td>
</tr>
<tr>
<td>Total Volume</td>
<td>50 &mu;l</td>
<td>25 &mu;l</td>
<td>25 &mu;l</td>
</tr>
<tr>
<td>MgCl2 conc.</td>
<td>unknown</td>
<td>2.5 mM</td>
<td>2 mM</td>
</tr>
<tr>
<td>dNTP conc.</td>
<td>each 0.2mM</td>
<td>each 0.2mM</td>
<td>each 0.2 mM</td>
</tr>
<tr>
<td>F primer conc.</td>
<td>0.1 &mu;M</td>
<td>0.2 &mu;M</td>
<td>0.2 &mu;M</td>
</tr>
<tr>
<td>R primer conc.</td>
<td>0.1 &mu;M</td>
<td>0.2 &mu;M</td>
<td>0.2 &mu;M</td>
</tr>
<tr>
<td>BSA conc.</td>
<td>0.3mg/ml</td>
<td>0</td>
<td>0</td>
</tr>
<tr>
<td>Taq conc.</td>
<td>1.25U/reaction</td>
<td>1.25U/reaction</td>
<td>1.25U/reaction</td>
</tr>
<tr>
<td>Template conc.</td>
<td>2 &mu;l/50 &mu;l</td>
<td>1 &mu;l/25 &mu;l</td>
<td>1 &mu;l/25 &mu;l</td>
</tr>
</tbody>
</table>

[STEPS]