Eccles:PCR Product Sequencing

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<div class=Section1>

<p class=MsoTitle><span style='font-size:10.0pt;font-family:Arial'>Sequencing Product Preparation</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<p class=MsoSubtitle><span style='font-size:10.0pt;font-family:Arial'>PCR clean up</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>2 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l PCR product</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'><u>4 </u></span><span style='font-size:10.0pt;font-family:Symbol'><u>u</u></span><span style='font-size:10.0pt;font-family:Arial'><u>l dH2O</u></span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>6 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l total (vary proportion based on PCR product conc’s)</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>to each tube add; 1/2 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size:10.0pt;font-family:Arial'>l exo (exonuclease)</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 

 1/2 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span

style='font-size:10.0pt;font-family:Arial'>l sap (shrimp alkaline phosphatase) </span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>put on Pre-seq rxn</span></p>

<ol style='margin-top:0cm' start=1 type=1> 

<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>15 min @ 37</span><span style='font-size:10.0pt;
    font-family:Symbol'>°</span><span style='font-size:10.0pt;font-family:
    Arial'>C</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>15 min @ 80</span><span style='font-size:10.0pt;
    font-family:Symbol'>°</span><span style='font-size:10.0pt;font-family:
    Arial'>C</span></li>

</ol>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>then into fridge to cool</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<h1><span style='font-size:10.0pt;font-family:Arial'>Sequencing reaction add</span></h1>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>0.64 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l primer</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>1 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l Big dye buffer</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>2 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l Big dye</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<h1><span style='font-size:10.0pt;font-family:Arial'>Sequencing reaction</span></h1>

<ol style='margin-top:0cm' start=1 type=1> 

<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>96</span><span style='font-size:10.0pt;font-family:
    Symbol'>°</span><span style='font-size:10.0pt;font-family:Arial'>C 1:15
    min</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>96</span><span style='font-size:10.0pt;font-family:
    Symbol'>°</span><span style='font-size:10.0pt;font-family:Arial'>C 45 sec</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>50</span><span style='font-size:10.0pt;font-family:
    Symbol'>°</span><span style='font-size:10.0pt;font-family:Arial'>C 45 sec</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>60</span><span style='font-size:10.0pt;font-family:
    Symbol'>°</span><span style='font-size:10.0pt;font-family:Arial'>C 3:30
    min</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>return to step 2 - 24x</span></li>

</ol>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<h1><span style='font-size:10.0pt;font-family:Arial'>Clean up</span></h1>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>2 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l 3M Sodium Acetate</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>10 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l dH2O</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'><u>50 </u></span><span style='font-size:10.0pt;font-family:Symbol'><u>u</u></span><span style='font-size:10.0pt;font-family:Arial'><u>l 95% ethanol</u></span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>62 </span><span style='font-size:10.0pt;font-family:Symbol'>u</span><span style='font-size: 10.0pt;font-family:Arial'>l per reaction tube</span></p>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<ol style='margin-top:0cm' start=1 type=1> 

<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>mix clean up solution with sample</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>let sit 15 min</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>20 min spin 4 000 rpm</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>decant and tap dry</span></li>
<li class=MsoNormal style='color:black;tab-stops:list 36.0pt'><span
    style='font-size:10.0pt;font-family:Arial;color:windowtext'>200 </span><span
    style='font-size:10.0pt;font-family:Symbol;color:windowtext'>m</span><span
    style='font-size:10.0pt;font-family:Arial;color:windowtext'>l 70% ethanol</span></li>
<li class=MsoNormal style='color:black;tab-stops:list 36.0pt'><span
    style='font-size:10.0pt;font-family:Arial;color:windowtext'>10 min spin 4
    000 rpm</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>decant and tap dry</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>repeat 5-7</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>set upside down on a clean dry towel until dry, (~ an
    hour)</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'> add 15 </span><span style='font-size:10.0pt;
    font-family:Symbol'>u</span><span style='font-size:10.0pt;font-family:
    Arial'>l high dye mix and re-suspend</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>95</span><span style='font-size:10.0pt;font-family:
    Symbol'>°</span><span style='font-size:10.0pt;font-family:Arial'>C for 2
    min and put on ice (burry the tubes)</span></li>
<li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt;
    font-family:Arial'>send to Alison Fitches with information on the run time
    needed (see table below)</span></li>

</ol>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&nbsp;</span></p>

<table border=1 cellspacing=0 cellpadding=0 align=left style='border-collapse: 

collapse;border:none'>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>Bases</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border:solid windowtext .5pt;
 border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>Time
 (min)</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>&lt; 200</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>75</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>200-250</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>80</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>251-290</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>85</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>291-320</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>90</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>321-350</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>95</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>351-380</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>100</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>381-410</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>110</span></p>
 </td>
</tr>
<tr>
 <td width=55 valign=top style='width:55.05pt;border:solid windowtext .5pt;
 border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>411+</span></p>
 </td>
 <td width=62 valign=top style='width:61.6pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'>120</span></p>
 </td>
</tr>

</table>

<p class=MsoNormal><span style='font-size:10.0pt;font-family:Arial'><br clear=ALL> </span></p>

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