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 was used. As template, the colony of interest was picked from a plate using a sterile toothpick and mixed into the reaction mixture. The cycling conditions are listed in table 1 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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Revision as of 22:43, 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 was used. As template, the colony of interest was picked from a plate using a sterile toothpick and mixed into the reaction mixture. The cycling conditions are listed in table 1 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
 
 
 

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