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-
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