DNA amplification but no bands on agarose gel, GC rich product, Roche
Posted 06 May 2009 - 04:30 AM
I've got a problem PCR. The product is 700bp, 77% GC rich promoter region, Tm of the product ~ 114. I'm, using the Roch GC rich kit, which has worked well for me in the past, using touch down or a modified slowdown PCR and deaz-GTP.
We ran the product on a 1% agarose gel and saw no bands, not even a smear. The DNA ladder works fine and shows up on the gel with good separation.
I measured the DNA conc of some of the PCR reactions and there is > 700 ng/ul DNA in each well. So amplification has occured. I originally thought that something the the Roche reagents (Buffer or GC rich resolution solution) may be holding the DNA back??? so we purified the PCR products using sodium acetate/ethanol precipitation (yeilds ~ 400-500ng/ul), and re-ran the samples on a 1% gel - still no bands. Loading buffer is used by all in the lab and works well and there is no evidence of leaking out the wells. Agarose and TBE buffer are brand new.
In silico PCR under less stringent conditions shows 2 possible products - 1) the expected amplicon and 2) ~ 70 kb product (not likely!)
Has anyone ever had similar problems? Does anyone know what might be going wrong? Or do you think a primer re-design needed?
Posted 06 May 2009 - 12:46 PM
In other words, I can see how a spec reading indicating that there's DNA in a sample could be completely wrong, but I can't see how there could be a good amount of DNA in a sample and it fail to stain with ethidium bromide. Thus, my conclusion would be the concentration estimate is wrong.
Posted 06 May 2009 - 04:50 PM
Posted 06 May 2009 - 05:47 PM
Can you tell us about the primers? How long are they, and what are their TMs? Do the primers have a non-template tail (RE sequence etc)? If so, don't include that sequence in the Tm calculations. Aim for a Tm of 65 - 70 C, just like any other PCR. It will be short, but as long as it's specific, it'll probably work.
I had to amplify an 87% gene, and my primers were only 15mers, so I know it works.
Posted 06 May 2009 - 10:13 PM
Also, what is the extension temperature of your Taq? Some Taq brands activate at 62oC not 70oC. Therefore, if you are running your annealing temperature above 60oC your primer may not get to bind properly before the Taq extension reaction begins.
Otherwise ditch the Roche stuff and go for good old DMSO (it can reduce the efficency of the Taq, so you may have to increase your Taq levels to compensate).
Posted 07 May 2009 - 07:15 PM