Team:SDU-Denmark/labwork/Protocols/PCR

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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols">mRNA Isolation</a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/PCR"><b>PCR</b></a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/Miniprep">Miniprep</a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/Checkdigest">Check Digest</a></td>
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<tr>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/3A">3A-Assembly</a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/colpcr">Colony-PCR </a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/Trans">Transformation</a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/gel">Gel-electrophoresis</a></td>
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</tr>
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<tr>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/revtrans">Reverse Transcriptase</a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/mutagen">Mutagenesis</a></td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/pcrgelclean">PCR-,gel clean-up</a></td>
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<h1>Polymerase Chain Reaction</h1>
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<h2>Proof-reading PCR using Phusion Hot Start II</h2>
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<img align="left" style="margin-bottom:0px; width: 755px; margin-top:-3px; padding:0;" src="https://static.igem.org/mediawiki/2011/2/21/Queens_CanadaTitleProtocols.png" usemap="#headermap" alt="Queen's">
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Component                50μl reaction              Final conc.
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-------------------------------------------------------------------
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H2O                      add to 50μl                -
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5x Phusion HF buffer      10μl                        1x
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10mMdNTPs                1μl                        200μM each
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Primer A                  xμl                        0,5μM
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Primer B                  xμl                        0,5μM
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Template DNA              xμl                        -
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(DMSO, optional)          (1,5μl)                    (3%)
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Phusion DNA Polymerase    0,5μl                      0,02U/μl
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</pre>
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</pre>
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</br>
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<b>PCR program: </b></br>
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<pre>95°C for 30 seconds
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<pre>25-35 cycles:
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98°C for 30 seconds
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Annealing temperature (5°C below primer melting temperature) 
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72°C for 15-30s/kb</pre>
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72°C for 5-10 minutes </br>
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4°C on hold</pre>
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</br>
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</br>
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<h2>Non-proof-reading PCR using Taq Polymerase</h2>
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</Br>
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5μl Dream Taq Buffer </br>
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5μl dNTP</br>
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1μl VF2</br>
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1μl VR</br>
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Udtag 10μl fra primer stock og fortynd i 90μl oprenset vand</br>
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1,5μl Dream Taq Polymerase</br>
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1μl Template</br>
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H2O up to 50μl </br>
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<b>PCR program: </b></br>
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<pre>95°C for 2 minutes
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols">mRNA Isolation      </a></span>    </regulartext>
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<pre>29 cycles:
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95°C for 1 minut 
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55°C for 30 sek
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72°C for 1min (1kb/min)</pre>
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<regulartext>
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72°C for 5 minutes </br>
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<classred1> <a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/PCR"><b>PCR</b></a></classred1>
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4°C on hold</pre>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/LiquidCulture">liquid culture      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/GlycerolStock">glycerol stock      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Miniprep">miniprep      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Digestion">digestion      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Ligation">ligation      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/GelElectrophoresis">electrophoresis      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/PCR">PCR      </a></span>    </regulartext>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Extraction"> extraction      </a></span>    </regulartext>
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<h2>Polymerase Chain Reaction</h2>
 
<!--
<!--

Latest revision as of 21:45, 26 September 2012

iGEM TEAM ::: SDU-DENMARK courtesy of NIAID


mRNA Isolation PCR Miniprep Check Digest
3A-Assembly Colony-PCR Transformation Gel-electrophoresis
Reverse Transcriptase Mutagenesis PCR-,gel clean-up

Polymerase Chain Reaction

Proof-reading PCR using Phusion Hot Start II


Component                 50μl reaction               Final conc.

-------------------------------------------------------------------
H2O                       add to 50μl                 -
5x Phusion HF buffer      10μl                        1x
10mMdNTPs                 1μl                         200μM each
Primer A                  xμl                         0,5μM
Primer B                  xμl                         0,5μM
Template DNA              xμl                         -
(DMSO, optional)          (1,5μl)                     (3%)
Phusion DNA Polymerase    0,5μl                       0,02U/μl

PCR program: 
95°C for 30 seconds 

25-35 cycles:
98°C for 30 seconds 
Annealing temperature (5°C below primer melting temperature)  
72°C for 15-30s/kb

72°C for 5-10 minutes 

4°C on hold


Non-proof-reading PCR using Taq Polymerase


5μl Dream Taq Buffer
5μl dNTP
1μl VF2
1μl VR
Udtag 10μl fra primer stock og fortynd i 90μl oprenset vand
1,5μl Dream Taq Polymerase
1μl Template
H2O up to 50μl

PCR program: 
95°C for 2 minutes 

29 cycles:
95°C for 1 minut   
55°C for 30 sek
72°C for 1min (1kb/min)

72°C for 5 minutes 

4°C on hold

Retrieved from "http://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/PCR"