I it seems that not picked up the right conditions for a partial denaturation of the fragments. Should try denaturant concentration gradient (urea) in a wide range as well as change the temperature (55-60C). Do you get a discrete fragment after PCR?

Thanks for the suggestion. I got what appeared to be a single bright band after PCR but it is possible that there are bands of lower intensities which I could not detect. I do use urea gradient at 60C.

Try using less PCR product, also check your PCR program. Probably, increasing the annealing temperature you could increase the quality of your amplicons. In my experience when you use touch down PCR programs, it always appears smears in your gel. Hope you can improve it, cheers

Thanks for all suggestions. I hope to improve my DGGE gels by modifying PCR conditions.

I am experimenting similar problems with my DGGE. Did you manage to solve it through PCR improvement (especially by increasing the annealing temperature)?

Hi Benjamin, Try using high fidelity enzymes like Phusion for PCR; definitely reduces non-specific bands. Good luck!

You could also try to add a 80 degree step in your PCR (after elongation) this could help to reduce primer dimer formation, I did that in G. Muyzer's lab. For your PCR: Do you use Primer for 16S? Your DNA is from biofilms? So the smear could also be an indication that you just have a really high diversity. But try to increase your annealing temperature, maybe add the 80 degree step, load different amounts of DNA on the gel and then have a look ;o) Good luck!

Thank you for your advices. Indeed, I solved the problem by increasing the annealing temperature. I could not find a better gradient than 50-65%, but other gradients revealed more smearous patterns. I am using bacterial 16S primers in sediment samples and the diversity seems realy high, with numerous and very close bands... I guess this can also partly explain the smears if the gradient is not optimized!

Yes, if you have samples with a really high diversity than you could get smear.

Hi Eileen, the additional 80 degree step that you've mentioned is for each pcr cycle, i'm i right? and how long it takes?