native vs denaturing gel for RPA

Shelley Cole shelley at DARWIN.SFBR.ORG
Tue Dec 10 13:28:11 EST 1996


Mike,
Some suggestions just from my own experience.

Make sure that your loading gel buffer has a final concentration of 1XTBE 
for your native gels. I do this by resuspending my samples in 2XTBE and 
then adding an equal volume of 2X loading buffer before loading.  
You can also try drying your native gels before exposing them to film.

Gel purify your probe on denaturing acrylamide gels only.

Shelley

Shelley Cole
Staff Scientist
SW Foundation for Biomedical Research
San Antonio, TX

On Tue, 10 Dec 1996, Mike Rott wrote:

> 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
> 
> 



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