This calculator generates a pipetting table for setting up PCR reactions with Thermo Scientific Phusion DNA Polymerases.

  1. Fill in the volume of template DNA you want to use, the number of PCR reactions you intend to run, and the individual reaction volume.
  2. Define if you would like to add some extra volume for pipetting. You can choose between an absolute volume, a number of extra reactions, or a percentage of the final volume.
  3. Fill in your stock concentrations and the desired final concentrations for primers, dNTPs, (DMSO), and Phusion DNA Polymerase.

The application will automatically calculate the pipetting volumes required for a premix and the volume of the premix needed for the final reaction.

Volume of DNA template/reaction µL  
 Extra volume
 Extra reaction
 % of volume
Number of reactions
Reaction volume µL
Total volume µL Extra volume%


 
Concentration
Volume (µL)
 
stock
 
final
 
per reaction
 
for premix
Phusion HF or CG buffer* X X  
Forward primer** µM µM  
Reverse primer** µM µM  
10 mM dNTPs mM mM  
(DMSO***, optional) % %  
Phusion DNA polymerase U/µL U/µL  

H2O  
Premix  
Template DNA  

*The HF buffer should be used as the default buffer for high-fidelity amplification. The GC buffer can improve the performance of Phusion DNA Polymerase on some difficult or long templates. More details can be found in the technical manuals for Phusion DNA Polymerases.

**The recommendation for final primer concentration is 0.5 µM, but it can be varied in a range of 0.2–1.0 µM, if needed.

***Addition of DMSO is recommended for GC-rich amplicons. DMSO is not recommended for amplicons with very low GC % or amplicons that are >20 kb.