Team:SDU-Denmark/labwork/Protocols/PCR

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<!-- //////////// Tabel \\\\\\\\\\ -->
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<td>Content</td>
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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>Content</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>Content</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>Content</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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<td>Content</td>
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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>Content</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>Content</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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<td>Content</td>
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<h2>Proof-reading PCR using Phusion Hot Start II</h2>
<h2>Proof-reading PCR using Phusion Hot Start II</h2>
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<img src="https://static.igem.org/mediawiki/2012/b/b4/Hot_Start.png" alt="some_text"/>
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<pre>
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<pre>
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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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<b>PCR program: </b></br>
<b>PCR program: </b></br>
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H2O up to 50μl </br>
H2O up to 50μl </br>
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<b>PCR-PROGRAM:</b> </br>
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<b>PCR program: </b></br>
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<pre>
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<pre>95°C for 2 minutes
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1. Start 95°C 2min </br>
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<pre>29 cycles:
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2. Denaturing 95°C 1min </br>
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95°C for 1 minut 
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3. Annealing 55°C 30sek</br>
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55°C for 30 sek
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4. Elonging 72°C 1min (1kb/min) </br>
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72°C for 1min (1kb/min)</pre>
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5. GO TO step 2, x 29 cycles</br>
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72°C for 5 minutes </br>
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6. End 72°C 5min</br>
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4°C on hold</pre>
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7. Hold 4°C infinite </br>
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</pre>
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<!--
<!--

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"