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primer design: end-point vs. RT


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#1 Mokuspok

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Posted 15 November 2004 - 07:43 AM

dear all,

this is the very first time that I'm designing RT primers :unsure: 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.

#2 biomed

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Posted 17 November 2004 - 12:39 PM

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




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