Before PAGE the PCRs were optimised with gradient PCRs and different Mg2+ concentrations according to results visualised on standard agarose gels, where we got one or two clear bands as expected (rarely some unspecific bands and sometimes primer dimers). With PAGE we want now enhance resolution.
Anyway when doing PAGE we never have the expected one or two bands, but either four bands, many bands or even a smear-like pattern. The same occurs with the size ladder (we use standard size ladders such as NEB's low molecular weight marker). The latter is recognised by the manufacturers and they suggest to use RNA loading dyes (i.e. denaturating conditions).
The question is now, why these weird band patterns occur in the samples and how to avoid it? Is it a PCR problem with badly designed primers (we got them from literature and have not much additional sequence information for the loci, but anyway when I checked them, the primer design was okay for me) and many unspecific products which are not separated in an agarose run. Or is it a problem that the PAGE conditions are not denaturating? Or other possible reasons (e.g. DNA binding proteins that interfere?)?
Thanks for any input and ideas...
Edited by hobglobin, 27 May 2013 - 09:22 AM.