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Team:Northwestern/Notebook/Protocols/PCR Amplification
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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. | + | 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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|align = "center" |Nuclease-free water | |align = "center" |Nuclease-free water | ||
| - | |align = "center" | | + | |align = "center" |To final volume (including mix) of 50 μL |
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|align = "center" |2X Phusion Master Mix | |align = "center" |2X Phusion Master Mix | ||
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| - | Transfer PCR tubes from ice to a PCR machine with the block preheated to 98 degrees C and begin thermocycling. | + | 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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Latest revision as of 19:29, 23 August 2011
PROJECT
RESULTS
CONSIDERATIONS
ABOUT US
NOTEBOOK
ATTRIBUTIONS
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
