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real time PCR primers


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#1 generider

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Posted 20 January 2005 - 07:30 PM

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!

#2 littlecell

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Posted 22 January 2005 - 10:14 AM

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
http://dorakmt.tripo...s/realtime.html
good look

#3 generider

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Posted 22 January 2005 - 03:39 PM

thanks
the website is very useful




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