milky gels, sequencer hint(?)

Robert Preston rapr at med.pitt.edu
Fri Dec 2 14:55:21 EST 1994


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).
None of the components (when tested) were at fault.  Since the gels ran OK
anyway, I ignored the opalescence. Then Shayan (ssharif at uoguelph.ca) saw
the post and told me the answer: when I cut the acryl:bis from 19:1 to
29:1, the opalescence disappeared.  It was the high bis amount that did it.

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.  I had thought the 19:1 seq stocks could not be the problem,
since the sequencing gels always looked fine.  BUT, they were 0.4 mm thick,
not 1.5 mm thick!

I presume that 0.4 mm thick sequencing gels (19:1) in fact ARE a bit
opalescent, but not enough to see.  This raises the possibility that
fluorescent signals from these gels run in a fluorescence-based automatic
sequencer may be degraded somewhat, relative to the signals that could be
obtained using a more transparent gel (i.e., one that has less bis).  

Possibly longer reads of sequence could be obtained, if the bands were 
optically sharper than they are with the 19:1 standard acryl:bis ratio.
This depends on how large the optical smearing might be, relative to the
limited resolution of the electrophoretic process itself. 

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

Mention me in the acknowledgements if this improves your sequencing runs.
First person to try lower Bis, let me know what happens.

-- 
Robert Preston                                    rapr at med.pitt.edu
U. Pittsburgh Pathology Dept.                   Pittsburgh PA 15261
vox 412-648-9573                                   fax 412-648-1916




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