|
POLYMERASE PRODUCT |
RECOMMENDED FINAL PRIMER CONCENTRATION (EACH) |
FINAL PRIMER CONCENTRATION RANGE (EACH) |
|---|---|---|
|
Q5®-XT Hot Start High-Fidelity 2X Mix (NEB #M2499) |
• Products under 1kb: 500 nM • Products over 1kb: 300 nM |
150-1000 Nm |
| Products over 8 kb may benefit from final concentrations between 150 and 250 nM. |
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.
Learn More
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.
