Team:Groningen/Template/MODULE/Notebook/protocols/PCR

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Polymerase Chain Reaction
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All PCR reactions were done with DreamTaq and Phusion polymerase from Thermo Scientific.
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DreamTaq polymerase
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For colony PCR DreamTaq polymerase is used. As the template, the colony of interest is picked from a plate using a sterile toothpick and mixed into the reaction mixture. The cycling conditions are listed in the table below.
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<th>Step</th><th>Temperature</th><th>Time</th><th>Number of cycles</th>
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<td>Initial denaturation</td><td>95 &deg;C</td><td>1 - 3 min</td><td>1</td>
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<td>Denaturation</td><td>95 &deg;C</td><td>30 sec</td><td rowspan="3">30</td>
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<td>Annealing</td><td>Tm - 5 &deg;C</td><td>30 sec</td>
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<td>Extension</td><td>72 &deg;C</td><td>1 min/kb</td>
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<td>Final extension</td><td>72 &deg;C</td><td>10 min/kb</td><td>1</td>
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Phusion polymerase
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Phusion polymerase is used when high fidelity is needed for the reaction. Phusion has been used for making of the BioBricks with PCR and generating specific mutations in genes with the use of primers. The cycling program is listed in the table below.
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<th>Step</th><th>Temperature</th><th>Time</th><th>Number of cycles</th>
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<td>Initial denaturation</td><td>98 &deg;C</td><td>30 sec</td><td>1</td>
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<td>Denaturation</td><td>98 &deg;C</td><td>10 sec</td><td rowspan="3">30</td>
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<td>Annealing</td><td>Tm - 5 &deg;C</td><td>30 sec</td>
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<td>Extension</td><td>72 &deg;C</td><td>15-30 sec/kb</td>
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<td>Final extension</td><td>72 &deg;C</td><td>10 min/kb</td><td>1</td>
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With difficult templates GC buffer with 1.5% DMSO was used.
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Latest revision as of 22:54, 17 October 2014

Polymerase Chain Reaction
All PCR reactions were done with DreamTaq and Phusion polymerase from Thermo Scientific.
DreamTaq polymerase
For colony PCR DreamTaq polymerase is used. As the template, the colony of interest is picked from a plate using a sterile toothpick and mixed into the reaction mixture. The cycling conditions are listed in the table below.
StepTemperatureTimeNumber of cycles
Initial denaturation95 °C1 - 3 min1
Denaturation95 °C30 sec30
AnnealingTm - 5 °C30 sec
Extension72 °C1 min/kb
Final extension72 °C10 min/kb1
Phusion polymerase
Phusion polymerase is used when high fidelity is needed for the reaction. Phusion has been used for making of the BioBricks with PCR and generating specific mutations in genes with the use of primers. The cycling program is listed in the table below.
StepTemperatureTimeNumber of cycles
Initial denaturation98 °C30 sec1
Denaturation98 °C10 sec30
AnnealingTm - 5 °C30 sec
Extension72 °C15-30 sec/kb
Final extension72 °C10 min/kb1
With difficult templates GC buffer with 1.5% DMSO was used.
 
 
 

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