The primers designed for the infusion cloning system have 15 bp overhang from the vector sequence and around 20 to 25 bp for the sequence from the gene of interest. The question is how to calculate annealing temperature for the pcr reaction in this case? We should consider complete primer sequence or the part which is exactly going to anneal in the initial pcr reaction for calculating annealing temperature? Or is there any way to increase the chances having better pcr amplification or any rules while setting pcr reaction?
in my poor experience, i have use primers with more than 20bp of overhang, and the best thing to do its put and annealing temperature two degrees below the tm; in my practical case my primer Tm (the annealing part) was 60ºC so my annealing temperature was 58ºC, after a few cycles i increase the annealing temperature to avoid the secundary structures common in huge primers.
the true is that the annealing part tm is the only one that matters, and after a few cycles it would doesn`t matter for the increases in the copies with the overhang part that will annealing. at the end you could have a few copies with errors, but the vast majority will be right
Dear Sir,
I got this information from the following link. Hope it will help you
The thumbrule for calculating the annealing temperature for a PCR primer is
Tm (°C) = 81.5 + 0.41(%GC) - (675/N)
where %GC is the percentage of G and C nucleotides in the oligo and N is the length of the oligo given in nucleotides.
Example calculation:
Oligo: 5'-TTAAAATGATAACCATCTCGC-3'
Tm = 81.5 + 0.41(33.3) - (675/21)
Tm = 81.5 + 13.7 - 32.1 (rounded to one decimal)
Tm = 63.1°C
When choosing the temperature for the PCR annealing step a good starting point is Tm - 5°C, for the above example that is 58°C.
https://jeltsch.org/annealing_temperature
Thanks, Dr. Artur for your answer. I agree with your suggestion as I found all methods useless with respect to calculating perfect Ta. I hope in near future someone will find a rigid solution for this so that we can save our time and resources. The only problem in using gradient pcr is it consumes more resources like enzymes which are very costly.
Dear Dr, Patil
Did you run the SOE PCR? Could you please share your experience in calculating the Tm?
when I think about it logically the whole primer sequence should be used to calculate annealing temperature, since only in the first cycle the overlapping part wont anneal.
Hi Yasaman,
Most of the genes are amplified well using the standard rule for calculating annealing temperatures considering only the overlapping primer sequence. For tricky genes, gradient pcr is the best way.
Thanks,
Suyash
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