I have synthesized a gene specific primer for amplification of my target gene. However, I am getting amplicon of only 100 bp rather than 210 bp (target amplicon size). So far I have tested so many optimization conditions for it but still getting non-specific band. I have cross checked the primer pair for non specific binding in my extracted DNA, it shows no complementary with any other region except for my target region.
Hi Moein,
which is the melting temperature (Tm) of your primers? Which are you using to amplify your target gene?
Tm is key for primer specificity. You should adjust the Tm so it is very close to the lowest primer Tm. I usually try to use 62ºC, although up to 55ºC should work.
Another chance is the appearance of primers dimers. An amplicon of 100bp could be the two primers dimerizing. If they are long enough, they could appear as primer dimers. You can use any on line tool to ceck for primer dimers, you can check OligoCalc, for instance.
Good luck
I think your gene of interest has a high GC content. so you can use GC buffer and for reducing secondary structures you should use DMSO (2% - 5%) during PCR. And try it with a low annealing temperature.
Moein Iranmanesh The melting temperature (Tm) of your primers is crucial for ensuring primer specificity. You should adjust the annealing temperature close to the Tm of the lowest primer, typically aiming for around 62°C, though temperatures as low as 55°C may also work. Primer dimers, which can form if the primers are long enough, could result in an amplicon around 100 bp in size. To avoid primer dimers, you can use online tools like OligoCalc to check for their presence.
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