RT PCR Standard curve amplification problem - (Oct/15/2007 )
Hi folks....I am having a problem amplifying my real time RT PCR standards. I am doing an absolute quantification curve using ds DNA. These standards have amplified pretty well in the past (a few weeks ago!) and now they didnt amplify The good news is my unknowns amplified on the same plate. I use the same mastermix, the same detectors, and of course the same primers and probes for my protein marker. I am quite confounded as to what is going on with my standards. Surely they couldn't have degraded, I mean all 10 of them?! Seems quite impossible, but I really dont know what else to suspect?! Any thoughts on what could be going on?
Are you sure that the heating block of your cycler is alright?