Taqman probe Tm ASAP! - (Mar/05/2007 )
My FAM-TAMRA Taqman probe Tm is 63C, length 26 bases. Primers Tm is 50C and 21 bases long. The product size is 130bp long. I'm using AmpliTaq Gold PCR Master mix in a Rotor Gene 3000. I use Dual-labeled probe setting with initial denaturation 95C 5min, and 2step cycling of 95C 20sec and 60C 1min for 40 cycles. Capture in FAM channel gain 5. BUT NO AMPLIFICATION CURVE!!! WHY???? I've repeated lots of times, but no avail. Is there any problem with the probe as the primers amplify well?
Is it because I'm using Amplitaq Gold master mix not *Taqman* Amplitaq Gold master mix?
I've read that primer Tms supposed to be 58-60C and probe Tm supposed to be 10C higher but what about mine and does it really matter? Thanks a lot, people, I'm at my wits' end and I don't want to but new probes...
taqmans are supposed to be validated before purchase. I would contact ABI for troubleshooting