Question: Designing primers for qPCR
0
gravatar for casley_queiroz
4.1 years ago by
casley_queiroz20 wrote:

When designing primers for qPCR, which sequence is better to consider, CDS or full gene?

gene • 2.8k views
ADD COMMENTlink modified 4.0 years ago by Biostar ♦♦ 20 • written 4.1 years ago by casley_queiroz20

Do you with full gene mean also intronic sequences, or CDS + UTR?

ADD REPLYlink written 4.1 years ago by WouterDeCoster44k

Just intron + exon, wihout UTR.

ADD REPLYlink written 4.1 years ago by casley_queiroz20

qPCR for cDNA I assume? Do you expect introns in your mature transcript/cDNA? Or are you talking about qPCR on genomic DNA?

ADD REPLYlink written 4.1 years ago by WouterDeCoster44k

Thank you for your support. I got an answer. Ideally designing between an exon + intron + exon. Thus eliminates the risk of losing a amplification if primers were designed in intro, because this would not be the cDNA.

ADD REPLYlink written 4.1 years ago by casley_queiroz20
1

Definitely not in an intron, best having intron spanning amplicon (one primer in exon n and the other in exon n+1) or a primer spanning the exon-exon junction. Are you sure you understand what you are doing?

ADD REPLYlink written 4.1 years ago by WouterDeCoster44k

I think I really understood, I drew one of the primers in the junction of two exon and the other within outher exon (exon 2 or 3 for exemplo).

ADD REPLYlink written 4.1 years ago by casley_queiroz20
1

Okay, I just came across this posts so this might be of interest for you: http://eu.idtdna.com/pages/decoded/decoded-articles/pcr-qpcr/decoded/2016/06/28/use-splice-junctions-to-your-advantage-in-qpcr

ADD REPLYlink modified 4.1 years ago • written 4.1 years ago by WouterDeCoster44k
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