1 reply to this topic

[Mokuspok]

dear all,

this is the very first time that I'm designing RT primers   and I'd like to find out what the main differences are between conventional and quantitative PCR primer design.

As of right now I have my target sequence (a catalytic site) in an exon, I have the neighboring exons too, so hopefully at least one primer will be spanning an exon-exon junction...

My questions are:
Do I decide if the probe is on the F or the R primer?
Do I use the online program provided by the company making my primers or do I hand-design them (for the end-point PCR I usually designed them manually)?
Is there anything else to look out for that I haven't thought about myself?

thank you so much.

[biomed]

Mokuspok, on Nov 15 2004, 08:43 AM, said:

dear all,

this is the very first time that I'm designing RT primers  and I'd like to find out what the main differences are between conventional and quantitative PCR primer design.

As of right now I have my target sequence (a catalytic site) in an exon, I have the neighboring exons too, so hopefully at least one primer will be spanning an exon-exon junction...

My questions are:
Do I decide if the probe is on the F or the R primer?
Do I use the online program provided by the company making my primers or do I hand-design them (for the end-point PCR I usually designed them manually)?
Is there anything else to look out for that I haven't thought about myself?

thank you so much.

Hi,
is the probe consist of fluoresence and quencher? Like the ABI probe? In this way , the probe should be target the fragment you are interested in, but not taged on either primer.
Biomed