DNA amplification but no bands on agarose gel, GC rich product, Roche - DNA amplification but no bands/smear in agarose gel (May/06/2009 )
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?
I would be vastly more inclined to believe that no amplification has occurred, because there are so many things that can throw off a spectrophotometric concentration estimate, but if there's DNA present in a sample at any appreciable concentration, EtBr staining will reveal it.
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.
A PCR reaction which has not been purified will always show a lot of DNA, since it has a very high concentration of dNTPs and primers, which will absorb UV in the same way as long DNA fragments.
Try a standard PCR, rather than the "high GC" kit, but add 1M betaine or 2-5% DMSO.
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.
When you did the spectro reading what did you use as your blank/ standard? You should use a blank that contains your reaction mix if you are using your PCR product directly (i.e. negative control).
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).
Blanking with the PCR reaction mix will not work well. The total number of nucleotides in the mix remains constant. All the PCR reaction does is rearrange them, so little change will occur after cycling. When purified to remove the dNTPs and excess primers, the product can be measured.