PAGE problems - (May/27/2013 )

We're doing PAGE with PCR samples using a quite large chamber (16 cm running distance) and non-denaturating conditions. Gel concentrations differ depending on expected band sizes (we expect one to two alleles per PCR, band sizes are 100-300 bp). The gels are cooled during the runs.
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...

have you monitored the temperature of the gel during the run? you may be melting the pcr products while they are traveling through the gel.

you can try a denaturing (urea) gel to determine if your pcr product is specific.

mdfenko on Tue May 28 12:00:42 2013 said:

didn't do that so far, but I'll check it, somewhere we had an infrared thermometer...
Is it enough to use a denaturing loading buffer or heating the sample or has to be the whole gel denaturing?
thanks.

if the whole gel isn't denaturing then you may find the sample renaturing during the run.

mdfenko on Wed May 29 14:21:14 2013 said:

okay then we try it with denaturating gels...
The temperature seems no problem as it is continuously around 25 degrees...until 50 degrees it should be no problem or even helpful to decrease running time...
Thanks mdfenko