Optimising Taqman probe - PCR is successful using SYBR but not with hydrolysis probe (May/26/2008 )
I have a PCR which I have optimised using SYBR green, and am now trying to do with a hydrolysis probe. (It is necessary to use a probe as I'm doing an allelic discrimination).
The SYBR reactions look fine - efficiency of 2, one specific product, no primer dimers. But when I do the reaction using a hydrolysis probe (self-designed, as am using genomic DNA) I run into problems. The replicate Cts are not as consistent as with SYBR - especially at limiting template concentrations, when some replicates don't give a signal at all (they amplified fine with SYBR), and the efficiency has gone up to 2.2 (which I didn't know could happen with a probe and no primer dimers!).
I wonder whether my probe Tm might be too low - it's only 64 degrees, 5 degrees higher than the primers. in other news, I'm using Roche reagents for their lightcycler 480 (the 384-well plate one).
With thanks for any comments,
There are many considerations that go into designing taqman probe, I hope you have done enough research on that. Many of my self-designed probes have worked like a charm, but some have created a similar trouble. It is not necessary for taqman to work, when SYBR has worked. You man even have to tweak PCR conditions in some resistant cases.
Here is some literature for you to read: http://search.vadlo.com/b/q?sn=158621799&a...probe&rel=0
See if you can troubleshoot it yourself, otherwise call Roche dudes.
thanks cell counter - I should have mentioned I designed the taqman probe properly, I've done several before and they've all worked fine. It's just this one that's a problem...
Sorry for my assumption. I guess the best thing to do in that case is to design another (if your sequence allows it), rather than troubleshooting this one. Well, let us see if somebody jumps in with some easy answer/s.