real time PCR primers-Real-Time PCR

Hi everyone, I'll start doing rt-pcr using sybr green chemistry and already ahve the primersPrimer 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.thanks!

-generider-

hi. 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

-littlecell-

thanks the website is very useful

-generider-

上一篇：real time sybergreen concentration-Real-Time PCR   下一篇：real-time PCR-Molecular Biology