qPCR negative controls

[Peter Ellis]

Gerardine.Persson from gmail.com wrote:
>
> My questions:
> - What exactly is actually amplified here? I've had many students ask
> me, and I grow tired of answering "I don't know, stop asking"

It'll vary from lab to lab depending on the source of the contamination.  It 
could be carry-over from your actual samples (and the CT will vary depending 
on the amount of carry-over), or it could just be primer dimer, in which 
case a melt curve will show you that it's a different amplicon from the one 
found in wells with template.

The only way to tell for sure what it is is to clone it and sequence it.  If 
it's the same sequence as the correct amplicon, then the answer is 
(unfortunately) that one or other of your reagents has been contaminated.

If you want to isolate the source of the contamination, you could run a 
series of different negatives leaving out different components of the 
reaction - leave out primer A, primer B or template in varying combinations. 
I'll bet dollars to donuts it'll be something like someone forgetting to 
change a pipette tip when setting things up, and thus transferring a 
fractional amount of template into either your primer aliquots, the enzyme 
mix itself, or even your water aliquot.

It *is* possible to get true negative controls, honestly!

Peter