native vs denaturing gel for RPA
rott at mailer.mpib-tuebingen.mpg.de
Tue Dec 10 10:06:38 EST 1996
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
thanks in advance,
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