milky gels, sequencer hint(?)
rapr at med.pitt.edu
Fri Dec 2 14:55:21 EST 1994
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
More information about the Methods