native vs denaturing gel for RPA

[Mike Rott]

I am new to RPAs so bought the Ambion kit. They recommend native gels,
supposed to give sharper bands and less problems with nicked hybrids.
However, I find that my denaturing gels give much sharper bands. Any
suggestions on improving native gel performance?

native gels are 6% acrylamide 19:1 in 1xTBE

loading buffer is 6% sucrose, sample are loaded directly

denaturing gel contains 7M urea, loading buffer is 95% formamide

samples are heated before loading

gels 0.4mm thick and run at 1500 volts for about and hour in a pharmacia
sequencing unit. Denaturing gels are run at 50 C and native gels at 18 C
using a recirculating water bath.

I have also had some problems with the probes getting stuck in the wells
during electrophoresis to gel purify the probe. So far only with native
gels.

thanks in advance,


mike