faint band of interest & primer-dimer prob - (Feb/28/2007 )

Hi all,

in my PCR, i m getting very faint positive bands with my sample and also a good band of primer-dimer. but with my positive control DNA, i always get a sharp band of my interest and almost no primer-dimer band. Faint band is giving too much problem in interpretation of results as primer-dimer size and band of interest (124bp) comes very near to each other in 2% gel.
so how to increase the intensity of band and reduce the primer-dimer? i ve tried increasing my amplification from 35 to 40 cycles. my reaction conditions r - 94-2min, 68-2min, 72-1 min and 72-10min. i m using IS6110 primer for M.tuberculosis.
Pls help.

regards
poonam

Try increasing template. I am expecting that more template will reduce primer-primer interactions in early cycles and will reduce your dimer band... Also why not run on a higher percentage gel?

I usually use DMSO to prevent unspecific primer binding and to get clean sharp bands. I suppose it may also work for primer-primer dimers, as it is also a form of non-specific binding. Try using 5% DMSO in your PCR mix.

Maybe increasing annealing temp may also help.

Good luck on that.

I would suggest using more DNA template for PCR and you could run your gel longer so that the PCR product seperates from the primer dimers.

Increasing cycles could potentially increase chances of mutations.

I don't see the rational of increasing the number of cycle. The normal 30 cycles will give you pretty much DNA. Even if you increase the number of cycle, but the taq polymerase is the limitation factor, hence you can't get more products.
In addition to increase your DNA template, I would suggest that you decrease your primers concentration, and yes, use DMSO in your reaction.
Good luck.

Thanx for all yr suggestions. i ll try using DMSO. but can someone explain me that why i get a sharp band with positive control DNA and faint/blurred band with test DNA? positive control DNA i dont isolate myself and received as a gift from Colorado State University and i think that might be purified DNA n thats why working fine. In my case i m using conventional Phenol-chloroform method for extraction.