Normally its a common scientific notion that as we decrease the annealing temperatur, it’s likely more primer dimers are formed. In this picture, I have observed something differen. As I increased the annealing temperature, primer dimer formation increased and exact amplicon size band became lighter
pcr: Gradient (extremely useful if you are unable to amplify the gene)
amplicon size: 968 bp
primer dimer: 200-300 bp
might be you are thinking why I am saying primer dimer at 200-300 bp site? I give you answe, first wanna listen fron you
Hi Muhammad Haroon
This is quite remarkable to find primer dimers of 200-300bp, may i know the size of your primers? What i feel here is that the 200-odd band is of some off-target amplicon.
Let me know,
Subham.
In my little knowledge, it's quite common that when annealing temperature is increased, the stringency of annealing is also increased leading to the specific and reproducible amplification. In normal practice keep the annealing temp low to to allow both forward and reverse primers to bind to the template.
Abdul Saboor, that why I am saying, primer dimer formation should decrease as annealing temperature is being inceeased
It's interesting to hear that increasing the annealing temperature resulted in increased primer dimer formation and lighter bands for the exact amplicon size. Usually, higher annealing temperatures lead to increased specificity by reducing nonspecific binding, which can lead to primer dimer formation. As for your observation of primer dimer formation at the 200-300 bp site, it's possible that your primers have some degree of complementarity, leading to non-specific binding and primer dimer formation. This could be exacerbated by the increased annealing temperature, which may allow for more stable primer-primer interactions.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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