1 reply to this topic

[Bill_Morris]

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

Edited by Bill_Morris, 16 November 2009 - 07:47 AM.

[gogreen]

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