PCR product multiple bands - (Sep/18/2006 )
I am trying to amplify the V3 region of the bacteria DNA using primers 518 r and 357 f with 40 nucleotides gc clamp. I got my samples from the sediment extracted using the Soil Master DNA Extraction Kit. My result is shown as attached below. Actually I tried several methods but the products are almost the same. My target product is really clear but I also got thin bands of around 300 bp and 400 + bp. I also have primer-dimers of less that 100 bp size. I tried several trials, but the result is the same. I even diluted my DNA sample to get rid of humic acid. I used several PCR conditions and used touchdown PCR. I even titrated my MgCl2.
Any suggestion on how can I improve next time. Actually, I’ll be doing a DGGE profile of bacterial communities. I am not sure if my product that I have now will be a good input for the DGGE gel… Any suggestion?
Thanks for your help…
what have you done to control the quality of your DNA preparation? also, have you tried adding less template?
To fix the primer dimer, check the 3' ends of each of your primers (say 5-10 bases), and make sure they don't complement the other primer, the primer itself, or other parts of your target sequence. If they do not match, you won't get primer dimer, and you might also clean up your reaction.
Aimikins. I just followed the protocol of the kit that I used. I diluted my DNA samples in steriled mili Q prior to PCR to get rid of some inhibitors.
Swanny, thanks for your suggestion.