taqman is giving initially raised then fall off amplification curve why? - changed template concentraion n baseline too.....but ? (Jul/18/2007 )
since pase few years i m doing this assay but suddenlly my taqman RT-qPCR (Mx3000) stratagene the amplification curve is getting raised initially and again falling down n making sigmoid curve along the base line..............
its happening not with a single set of probe but with 3-4 probes different seys that means its not the fault of probe.......i changed mastermix n RNA samples too.............still the things are unanswered.
my 3 yeras experience suggested me to optimize the template concentration i did that but stil same pattern i m getting..........
plz suggest me the parameters to be followed.........i use 0.5ul of RNA thats enough for amplification and was working nicelly.
can anybody tell me the solution or its a instrument fault.....................if somebody suggest the lamp problem theni will say out of 2000 Hrs only 1000Hrs are completed in my machine means lietrally it shud not be the prob.
I would be inclined to think that you are running out of a reagent in the middle of your reaction, something that you're using in every well. even if you make new mastermix, perhaps this could be the problem. for example, if your new mastermix is made from a newer stock of dNTPs that wasn't diluted properly or had degraded, then you would see something like this.
I would also consider checking the heating block of your cycler.