GSP Gene Specific Primers for Real Time PCR

[dnamolecule]

Hiya all,

I am looking to create gene-specific primers (GSP) for my real time PCR, of a gene which spans exon-to-exon boundaries.

Im going to be using a qRT-PCR one-step kit with SYBR dyes.

How can I design my own gene specific primers for my gene for real time PCR?

Do most of you buy your primers pre-made?


thanks!

[danfive]

Acquire the expressed gene sequence, copy and paste it in primer design software (many online like ---> http://www.idtdna.com/Scitools/Appli...s/Primerquest/ ) then pick a pair from potential matches that come up. You will have to experimentally determine which pair is most specific and what its optimal PCR conditions are, you should do melting curve analysis (have a relatively pure PCR positive control) or agarose gel to check for nonspecific products. Your will usually order your primers by specifying the sequence and concentration you want and receive them in lyophilized form. From here you can dissolve them at a concentrated stock solutions like 500X, then make working dilutions as needed at 50X, with your final PCR concentration for each primer being 1X.