My supervisor always tells me that no matter what the sequence of the primers is the optimal PCR annealing temperature is 55°C. Take with that knowledge what you will.

Dear Muhammad

the most of books report " generally you should use an annealing temperature about 5°C below the T m of your primers"

However in my experience, the theoretical Tm can change a lot on the basis of the software that you use for the calculation and on the polimerase buffer.

For example for PIPE cloning i'm using a lot the Kapa Hifi master mix polimerase and work well with high annealing temperatures (often to obtain a good result i have to use Ta = TM + 5 or more, while other taqs as Q5, Pfu ultra fusion II, Clone Amp work well at lower Ta.

In you case i think you can start a first attempt with 60°C and see whats happen or in case you have a PCR machine that can run in parallel more Ta, direclty perform a trials with 3 different Ta (55-60-65) for example.

good luck

Manuele

The annealing temperature (Ta) chosen for PCR relies directly on the length and composition of the primers. Generally, you should use an annealing temperature about 5°C below the Tm of your primers.

You can use this online tool to calculate Ta of your primers https://www.thermofisher.com/sg/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html Good luck Muhammad Jawad

I would start from 55-60 first. My supervisor always suggests trying 58 for annealing. Every single time it worked no matter how big is my primer. It might work for you. I have primers having 55 bp in length and 10 nucleotides overhang and Tm around 72. I used 58 and it worked.

For your case, I would also like to know the GC% of your primers. Give it a try with 58 first. Rules do not work always.

You can calculate it on the following page: https://tmcalculator.neb.com/#!/main

Beyond the result it gives you, the best way to estimate the anneling temperature is empirical. A good start can be with an anneling temperature 2-5 degrees below the Tm of your primer with lower Tm.

All annealing tempratues work well ranging from 56 to 66C. Unless/Until your fragment have specific conditions (i.e repeats etc). For that case sometimes you may have jump as higher as 72C and lower to 48C. “Veriflex Step” can be used to optimize temptature for saving time.

Use one of the free online calculators to get a starting temperature. If you really want to optimize your reactions, then set up a gradient PCR with annealing temps above and below that calculated temperature. Really robust primer pairs will work well in a wide range of temperatures (48-65).