milky gels, sequencer hint(?)

Song Tan tan at mol.biol.ethz.ch
Thu Dec 8 08:15:21 EST 1994


In article <rapr-0212941455210001 at quadra800.pathology.pitt.edu>,
rapr at med.pitt.edu (Robert Preston) wrote:

> Netfolks:
> 
> Recently I posted asking for an expanation of the appearance of a milky
> opalescence in my PAGE gels (8%, non-denaturing, 0.5x TBE, 1.5 mm thick).

[snip]

> I checked back in my notes: sure enough, I'd been previously using a
> 29:1 mix in the gels that weren't milky before.  The problem came up
> when I tried to use premixed 19:1 sequencing stocks instead of my previous
> custom mix.  

[snip]

> Who set the 19:1 ratio, anyway?  Does anyone know why 29:1 could not be used
> for sequencing gels?

My guess is that the 19:1 ratio was chosen for use with 6-8% acrylamide
sequencing gels to increase the physical strength of the gel.  Higher
ratios of bis make very low concentration acrylamide gels sturdier (i.e.
less gooey) and easier to handle.  However, high ratios of bis like 19:1
used with high concentration acrylamide gel (12% acrylamide or higher)
cause the gel to become more brittle and much more likely to crack when
dried under a vacuum.

I can't think of any reason why you can't use 29:1 bis:acrylamide for your
8% sequencing gels especially since you're getting good results with
them.  For the reasons indicated above, I prefer bis ratios of 29:1 or
39:1 when I run >12% acrylamide sequencing type gels for footprinting.

For what it's worth, I've also seen milky stacking gels in my SDS-PAGE
gels recently.  In this case, I don't think it's the bis ratio causing the
milkiness, but I haven't nailed down what the cause is either.  Hasn't
affected the quality of the gels, so I haven't spent the time trying to
track it down.

-- 
Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email:  tan at mol.biol.ethz.ch



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