FAQ: What should the final primer concentration be in my PCR?

POLYMERASE PRODUCTS

RECOMMENDED FINAL PRIMER CONCENTRATION (EACH)

FINAL PRIMER CONCENTRATION RANGE (EACH)

Q5® Polymerases

500 nM

200-1000 nM

Phusion® Polymerases

500 nM

200-1000 nM

OneTaq® Polymerases

200 nM

50-1000 nM

Taq Polymerases

200 nM

50-1000 nM

Hemo KlenTaq

300 nM

50-1000 nM

LongAmp® Polymerases

400 nM

50-1000 nM

We recommend a final primer concentration of 500 nM when using Q5 and Phusion polymerases due to their high 3´-5´ exonuclease activity.
 
We recommend a final primer concentration of 200 nM when using Taq-based polymerases, including OneTaq and EpiMark®.
 
Please refer to specific protocols on the product pages for details regarding range recommendations.
 

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Archaeal Family B polymerases, such as Q5 and Phusion, possess strong 3´-5´ exonuclease activity, which can digest nucleotides on the 3' end of primers and increase the likelihood of nonspecific amplification. Relative to Family A polymerases like Taq and OneTaq, a higher final primer concentration overcomes these effects and promotes specific product formation.
 
Generally, efficient PCR requires optimal ratios of magnesium, other ions, and primers to promote specificity by stabilizing the negative charges on the phosphate backbone. While the composition of Q5 and Phusion DNA Polymerases are proprietary, their buffers are optimized for using a final primer concentration of 500nM.