Team:Northwestern/Notebook/Protocols/PCR Amplification

From 2011.igem.org

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Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98 degrees C). All components should be mixed and centrifuged prior to use. It is important to add Phusion Master Mix last in order to prevent any primer degredation.
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Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98 degrees C). All components should be mixed and centrifuged prior to use. It is important to add Phusion Master Mix last in order to prevent any primer degredation. For 50 uL reaction:
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For 50 uL reaction:
 
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Transfer PCR tubes from ice to a PCR machine with the block preheated to 98 degrees C and begin thermocycling.  
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Transfer PCR tubes from ice to a PCR machine with the block preheated to 98 degrees C and begin thermocycling. Thermocycing conditions for a routine PCR:
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Thermocycing conditions for a routine PCR:
 
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Revision as of 19:29, 23 August 2011

RETURN TO IGEM 2010


PCR Amplification


We used [http://www.neb.com/nebecomm/products/productM0531.asp Phusion High Fidelity PCR Master Mix] from New England Biolabs.


Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98 degrees C). All components should be mixed and centrifuged prior to use. It is important to add Phusion Master Mix last in order to prevent any primer degredation. For 50 uL reaction:


10 uM forward primer 2.5 uL
10 uM reverse primer 2.5 μL
Template DNA 1 uL
Nuclease-free water to final volume (including mix) of 50 μL
2X Phusion Master Mix 25 μL


Transfer PCR tubes from ice to a PCR machine with the block preheated to 98 degrees C and begin thermocycling. Thermocycing conditions for a routine PCR:


Initial denaturation 98 degrees C 30 seconds
25-35 cycles 98 degrees C 5-10 seconds
45-72 degrees C 10-30 seconds
72 degrees C 15-30 seconds
Final extension 72 degrees C 5-10 minutes
Hold 3 degrees C