denaturing PAGE question

brett brett at BORCIM.WUSTL.EDU
Tue Jun 3 08:28:40 EST 1997


>I know that sequencing gels are run 'hot' (50-60 degrees)  but should a
>thicker (0.75 mm) denaturing PAGE gel (e.g. set up in a BioRad Protean IIx
>apparatus), used for resolving radioactive PCR products in the 100-500 bp
>range in my case, also be run hot?  And do samples need to be denatured by
>heat if they're mixed with formamide-containing gel loading buffer?
>
>
>Sambrook et al. only has protocols for olignucleotide gels, which is why
>I'm asking.  

If you run your gel such, it is unlikely you will resolve in the 100-500 bp
range. Maybe 100-500 *nt* depending on your %gel, but it doesn't sound as
though that is what you want. I use the mini-protean system all the time for
low mw separations, such as you desire. I'd recommend just pouring a 5% gel
in 1xTBE (no urea!), and running at 25mA. No need for formamide (denaturant),
but I have found glycerol-based sample buffer seems to work better than Ficoll
buffers on these gels. Staining takes <5 minutes in 1-10ug/ml EtBr and bands
are very sharp. If it starts to heat or band edges are smeary, turn it down.
Good luck.
Brett Lindenbach
Dept Molecular Microbiology
Washington University School of Medicine
St Louis, MO

"One thousand miles an hour, I'm just like anyone.
I want to feel the road of tar beneath the wheel
named extinction" - Pixies




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