RT-PCR primer design - RT-PCR primer design...need ur help (Oct/24/2008 )

Hey guys,
Can someone tell me how to design a RT-PCR primer?
I am the first one(first time) to do it in my lab.
I got a couple of informations from the net but some are
just to difficult to understand for a beginner like me.
What is the most important factor (Max score, Total score
,E-value...) to look when you blast the amplicon sequence
(or the primers)against genome database.
Thank you for your help.

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Hi!
I usually use Primer Express, a software of Applied Biosystem. The most important value is the totral score, which is a sum of all the penalty (like Tm or amplicon langht). In general the most important rules are the following:
1) Tm must be >60°C (most of qPCR thermal profile have a 60°C anneling/extension step)
2) your amplicon must be small (usually 100-200 bp)
3) if it's possible you must design two exon overlapping primers, if you use sybr green.
Anyway there are qPCR primer database available on web (where you can find qPCR primer sequence of most common genes).

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You could learn it all from scratch - and I'd recommend that. However if you want to quickly get this up and running buy some commercial primers from someone like Qiagen.

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thanks so much

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