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<h1 class="sectionedit1"><a name="fusion_pcr" id="fusion_pcr">Fusion PCR</a></h1>

<h3>PCR mix I</h3>
<table class="tabelle">
<tr><th><center>ingredient</center></th><th><center>volume</center></th></tr>
<tr><td><center>Template</center></td><td><center>200 - 300 ng</center></td></tr>
<tr><td><center>Q5 Polymerase</center></td><td><center>0.5 µl</center></td></tr>
<tr><td><center>dNTPs</center></td><td><center>4 µl</center></td></tr>
<tr><td><center>DMSO</center></td><td><center>1 µl</center></td></tr>
<tr><td><center>Q5 reaction buffer (5x)</center></td><td><center>10 µl</center></td></tr>
<tr><td><center>dH<sub>2</sub>O</center></td><td><center>up to 45 µl</center></td></tr>
</table>

<h3>PCR run I</h3>
<table class="tabelle">
<tr><th><center>temperature</center></th><th><center>time</center></th></tr>
<tr><td><center>98 °C</center></td><td><center>5 min</center></td></tr>
<tr><td><center>98 °C</center></td><td><center>30 s</center></td></tr>
<tr><td><center>** °C</center></td><td><center>30 s</center></td></tr>
<tr><td><center>72 °C</center></td><td><center>35 s per 1 kb amplicon length</center></td></tr>
</table>
<p>Repeat steps 2 till 4 for 5 times.</p>
<p>** Calculated annealing temperature of the whole owerlap (OligoCalc, basic). If the temperature of step 3 is close to or higher than 72 °C, a touchdown PCR should be performed: The temperature of step 3 will be reduced for 0.5 °C every cycle, starting at 72 °C.</p>

<h3>PCR run II</h3>
<p>Add 2.5 μl of each primer that anneal at the ends of the new construct.</p>
<table class="tabelle">
<tr><th><center>temperature</center></th><th><center>time</center></th></tr>
<tr><td><center>98 °C</center></td><td><center>5 min</center></td></tr>
<tr><td><center>98 °C</center></td><td><center>30 s</center></td></tr>
<tr><td><center>63 °C</center></td><td><center>30 s</center></td></tr>
<tr><td><center>72 °C</center></td><td><center>35 s per 1 kb amplicon length</center></td></tr>
<tr><td><center>72 °C</center></td><td><center>10 min</center></td></tr>
</table>
<p>Repeat steps 2 till 4 for 18 times.</p>

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