I have a pair of primers designed for a site-directed mutagenesis experiment. The Tm of the forward primer is 74.5 °C, and the Tm of the reverse primer is 70.5 °C. For amplification, I am using Phusion High-Fidelity PCR Master Mix with HF Buffer.
Kindly let me know the appropriate range of annealing temperatures to test in a gradient PCR to determine the optimal annealing temperature for these primers.
Annealing temperatures above 70 °C seem way too high to me, maybe consider redesigning your primers. If you insist, I would try running your PCR across a gradient from about 66 °C to 78 °C. This twelve‐degree span brackets each primer’s Tₘ by roughly ±6 °C, ensuring that you sample the 3–5 °C below each Tₘ—where annealing is most specific.
hi
yes I agree with Kristyna
but if you can't design new primers, test a 2-step PCR protocol as following:
https://www.neb.com/en/protocols/2013/01/11/pcr-optimization-e0555?srsltid=AfmBOorN4neEgAmEdiKrOC3sBa8GlowwLHCn8Axxo8gerp3PAuPxOLHo
best
fred
Tm and annealing temp are not equivalent. You want the annealing temp to be lower than the Tm. Lots of online calculators you could try or you can just wing-in and try a big range (old-school).
Just pick a range that goes above & below. I've done 48*C-72*C, but you could probably start at a higher temp since the Tm is pretty high.
The optimal annealing temperature should be 5-8°C below the calculated Tm (when greater than 68°C). For best results the Tm and length of both primers should be equal. First: Calculate the annealing temperature for both primers: Tm-(5to8°C) and take the average. Second: the range for the temperature in a gradient PCR: average calculated annealing temperature +- 5 °C. Third: the optimal annealing temperature is the temperature with the most amplified PCR product.
Link:
https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/pcr/ introduction/pcr-primer-design
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