I have a primer where the G% of forward primer is 50% and that of reverse is 20% , and annealing temperature is 48 degree C ,what could be the reason of not getting pcr product? can GC% really matter or i need to keep in changing annealing temperature?
possible reasons are secondary structure ( hairpin or G quadruplex) on the primer or as the primer is GC rich perhaps the template is also GC rich and has secondary structure issues. Try a temperature gradient pcr with DMSO or 1M betaine in the reaction mix and if that fails design primers that have a higher annealing temperature
Hi Amiti,
designing primers need to follow some rules and one of them is that Tm difference between primers must be bellow 2°c, defining the Tm you'll use for PCR (it allows therefore annealing of both primers at same time/temerature during the cycles).
if the Tm of one of them is too high or too low compared to the Tm of the PCR, you'll miss some annealing and PCR product you won't have.
fred
I forgot one thing, more you'll have GC and more the Tm will be high (long time ago we calculated the Tm as Tm= 4x(number of G/C) + 2x(number of A/T). but long time ago......
I use Oligo7 but you can also use some free tools as (https://www.thermofisher.com/fr/fr/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html) or (https://tmcalculator.neb.com/#!/main)
fred
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