NTC with specific amplification - (Nov/16/2009 )

OK - firstly excuse the fact that this topic is posted on here a million times and i have read most of them. However i need to bounce ideas of someone (anyone) else who knows what they are talking about.

Right - Im doing qRTPCR in a BioRad miniopticon using the BioRad iScript sybr green master mix.

I have been getting amplification in the NTC. At first i thought that it was likely to be primer dimer as i had seen it previously on Gels so i redesigned the primers and now don't see any primer dimer either on gels or in the melt curve (So no primer dimers)

I see a single band of about the same intensity as i do on the positive control at exactly the same size. Also they have identical melt curves. (so its a specific amplification)

So the components of the reaction are the master mix, water and primers. I have changed all three and still the amplification. I did however see a drop from a ct of about 25 to 30-32ish. Meaning that some/all of those components must have been contaminated. (so have now take both my class two hood and pipettes to pieces and cleaned with ethanol/an antimicrobial spray and RNAaway spay. I have introduced new filter tips, new tubes etc. I have even changed the tape used to seal the also new plates that the reactions run in. STILL I GET THE SAME BAND.

The only thing i have possibly got left (and it seems pretty remote) is that when i opened my stocks of primers i introduced the contamination into the stocks either via the pipettes or from the hood.

Can anyone think of any other possibility.

Thanks in advance for the ideas

If you doubt the specific primer, if I were you, I would run PCR with some other primer which I know was working well and use fresh reagents, clean pipettes and filter tips as u said...since you changed almost all the other parameters, there is nothing else to go wrong..I had a similar problem some time ago and the primers were the culprits in that case..

all the best