Team:Cambridge/PCRprotocol

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PCR using Phusion DNA polymerase:

If this reaction is to be done from scratch, add the reagents in the order they are listed in the table. When multiple PCR experiments need to be run concurrently, everything that is not experiment specific (primers and template DNA) can be made up ahead of time as a master mix. In this case the master mix should be kept on ice as it contains enzymes. The primers and template DNA should be added to a PCR tube and the master mix added just before the sample is put in just before PCR begins.


Reagent50 µl reaction20 µl reactionFinal Concentration
H2OAdd to make 50 µlAdd to make 20 µl
5x Phusion buffer10 µl4 µl1x
10 mM dNTPs1 µl0.4 µl200 µM
Primer 1x µlx µl0.5 µM
Primer 2x µlx µl0.5 µM
Template DNAx µlx µl
Phusion DNA polymerase0.5 µl0.2 µl0.02 u/ µl

N.B. The volume of primer that needs to be added will vary in accordance with the concentration of DNA in the initial sample


PCR machine settings:

Temperature (oc)Time (s)
Hold 195360
Cycle (30x)Denaturing9810
Annealing6030
Elongation72120




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