real time PCR primers-Real-Time PCR

互联网2013-12-26

2305

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.
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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