Difference between revisions of "Team:Freiburg/Protocols/PCR"

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<h3 class="sectionedit2"><a name="programm_normale_pcr" id="programm_normale_pcr">Programm normale PCR</a></h3>
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<p>
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<h3 class="sectionedit2"><a name="programm_normale_pcr" id="programm_normale_pcr">Standard PCR Programm</a></h3>
 
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<div class="level3">
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<p>
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For amplification of different DNA fragments from plasmid templates, various PCR approaches were used. In general, PCRs were performed with Phusion High Fidelity DNA Polymerase (NEB). The components of the reaction mixture were used in defined amounts/concentrations recommended by the manufacturer.
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</p>
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<p>
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<body>
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<h3>PCR mix</h3>
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<table class="tabelle">
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<tr><th><center>ingredient</center></th><th><center>volume</center></th></tr>
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<tr><td><center>Template</center></td><td><center>200 - 300 ng</center></td></tr>
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<tr><td><center>Primer (10 µM)</center></td><td><center>1 µl each</center></td></tr>
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<tr><td><center>Q5 Polymerase</center></td><td><center>0.5 µl</center></td></tr>
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<tr><td><center>dNTPs</center></td><td><center>4 µl</center></td></tr>
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<tr><td><center>DMSO</center></td><td><center>1 µl</center></td></tr>
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<tr><td><center>Q5 reaction buffer (5x)</center></td><td><center>10 µl</center></td></tr>
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<tr><td><center>dH<sub>2</sub>O</center></td><td><center>up to 50 µl</center></td></tr>
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</table>
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Add GC enhancer if the GC content of the template [??] is very high.
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</body>
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</p>
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<p>Primers for DNA amplification were designed to have a melting temperature of about 60°C (detailed information can be found in the Oligo list). The following cycling conditions have been used for standard PCRs. Extention times were calculated according to NEB's recommendations for Phusion High-Fidelity Polymerase.
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<td class="col0 leftalign"> 98°C  </td><td class="col1 leftalign"> 5 min        </td><td class="col2 leftalign"> Denaturierung   </td>
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<td class="col0 leftalign"> 98°C  </td><td class="col1 leftalign"> 5 min        </td><td class="col2 leftalign"> Initial denaturation   </td>
 
</tr>
 
</tr>
 
<tr class="row1">
 
<tr class="row1">
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<th class="col0 leftalign"> 98°C  </th><th class="col1 leftalign"> 30 s        </th><th class="col2 leftalign"> Denaturierung   </th>
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<th class="col0 leftalign"> 98°C  </th><th class="col1 leftalign"> 30 s        </th><th class="col2 leftalign"> Denaturation   </th>
 
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<th class="col0 leftalign"> 72°C  </th><th class="col1 leftalign"> 30 - 40 s pro kb  </th><th class="col2 leftalign"> Elongation     </th>
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<th class="col0 leftalign"> 72°C  </th><th class="col1 leftalign"> 30 - 40 s pro kb  </th><th class="col2 leftalign"> Extention     </th>
 
</tr>
 
</tr>
 
<tr class="row4">
 
<tr class="row4">
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<td class="col0 leftalign"> 72°C  </td><td class="col1 leftalign"> 10 min        </td><td class="col2 leftalign"> Restelongation </td>
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<td class="col0 leftalign"> 72°C  </td><td class="col1 leftalign"> 10 min        </td><td class="col2 leftalign"> Final extention </td>
 
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<body>
 
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<h3>PCR mix</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>Primer (10 µM)</center></td><td><center>1 µl each</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 50 µl</center></td></tr>
 
−
</table>
 
−
Add GC enhancer if the GC content of the template [??] is very high.
 
−
</body>
 
−
 
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</p>
 
 
<div class="tags"><span>
 
<div class="tags"><span>
 
<a href="/igem2015/doku.php?id=tag:protocols_old&amp;do=showtag&amp;tag=protocols_old" class="wikilink1" title="tag:protocols_old" rel="tag">protocols old</a>
 
<a href="/igem2015/doku.php?id=tag:protocols_old&amp;do=showtag&amp;tag=protocols_old" class="wikilink1" title="tag:protocols_old" rel="tag">protocols old</a>

Revision as of 14:47, 16 September 2015

""

PCR

Standard PCR Programm

For amplification of different DNA fragments from plasmid templates, various PCR approaches were used. In general, PCRs were performed with Phusion High Fidelity DNA Polymerase (NEB). The components of the reaction mixture were used in defined amounts/concentrations recommended by the manufacturer.

PCR mix

ingredient
volume
Template
200 - 300 ng
Primer (10 µM)
1 µl each
Q5 Polymerase
0.5 µl
dNTPs
4 µl
DMSO
1 µl
Q5 reaction buffer (5x)
10 µl
dH2O
up to 50 µl
Add GC enhancer if the GC content of the template [??] is very high.

Primers for DNA amplification were designed to have a melting temperature of about 60°C (detailed information can be found in the Oligo list). The following cycling conditions have been used for standard PCRs. Extention times were calculated according to NEB's recommendations for Phusion High-Fidelity Polymerase.

Lid temperature: 98°C

98°C 5 min Initial denaturation
98°C 30 s Denaturation
63°C 30 s Annealing
72°C 30 - 40 s pro kb Extention
72°C 10 min Final extention
4°C hold

18 - 25 Zyklen (Schritt 2-4)