Taqman rtPCR primer and probe design - (Mar/17/2009 )
I have been attempting to design qPCR primers using Perlprimer. Unfortunately, this programme does not allow you to search for an appropriate Taqman probe for the corresponding sequence (cDNA) as part of a real-time PCR quantification (or is there a way?).
Our lab has no licence to the Beacon designer tool, so I have tried autoprime instead, requesting an internal hybridisation oligo (presumably a sequence which I can use as basis for the Taqman probe) as part of the search.
I would appreciate if someone could confirm the appropriate parameters for me. So far I have left all the settings unchanged, other than narrowing the left and right primers Tm to a range within 1 degreeC from one another, and defining the amplicon to be 200-350bp. Autoprime gives me appropriate primers for my sequence, but most the time the internal hybridisation oligo overlaps one of the two primer sequences, mostly that of the right primer, which surely cannot work as a probe.
I would be very grateful for any other advice on how to design the primers and the probe, preferrably with a free software/online tool available and the recommended settings/parameters (oligo size, Tm, position) I should use.
Primer3 can design both primers and probe, but I never used it for that.
My lab has software for this.
I did a short search in google and this is what I found:
GenScript, a real time primer database, biosearchtech free after registration, Beacon Designer™ Free Edition, RealTimeDesign free after registration, AlleleID
Keep in mind, I haven't tried it out so I can't vouch for the results
Roche Applied Sciences also has a good online program for picking a probe from their universal probe library.