Protocol Online logo
Top : Forum Archives: : Real-Time PCR

real time PCR primers - (Jan/20/2005 )

Hi everyone, I'll start doing rt-pcr using sybr green chemistry and already ahve the primers
Primer design tips for real time suggest Tm to be 58-60 or at least over 50.
Why is that? How important is that?
My Tm's are all around 53 and I guess that in any case I can set the temperature in the annealing step of the PCR to fit my primers.
Question 2: Tm's in my designing software are different than those provided by the synthesis company... who should I trust?
Any advise will be more than welcomed.


i think the high-Tm primers will have more specific product and decrease the possibility to generate dimers. and as for the exact Tm, you just use the Tm=4xG/C number +2xA/T number, because your primers is about 20-28bp.
i recommend your read the data at
good look


the website is very useful