Non-specific Real time PCR signals - (Nov/12/2007 )

Hi everyone!

I was wondering if anybody could help me? I'm at my wits end with a real time PCR that will not behave. I'm running a Taqman based assay over a period of 40 cycles. For my target bug, it's wonderful. However, I'm now testing against genetic near neighbour organisms. From the genetic data available on line, there should be no cross reaction. Indeed when testing small values such as 10pg- 100fg, there is no problem at all. However, when I tested against more concentrated samples (5ng +), I get positive values at around 36 cycles (corresponding to 10fg of my target). I've tried changing the annealing temperature (increasing all the way to 68oC) and changed the MgCl2 concentration to hopefully make the reaction more specific. However, this has been of no use. I've tried Southern Blotting of the extracts to see if the target is present, but this has proved unsuccessful. I thought that our samples were contaminated so I've got fresh DNA from a lab that has never isolated my target bug. However, this too is amplifying quite well .
I feel like I'm going round in circles and I know that the easiest way out is to cut at 35 cycles but I want to know what is going on!!! I've digested the DNA (with EcoRV) and used DMSO to see if it was a structural thing, but that has failed too! Can anyone explain what is going on or is it that the gods of PCR are angry with me!

Many thanks!

Stand free 1983

http://www.protocol-online.org/forums/inde...mp;#entry117029