mkennedy at chmeds.ac.nz
Wed Mar 30 19:37:56 EST 1994
In article <2n2ha1$39c at quartz.ucs.ualberta.ca>, asimmond at gpu.srv.ualberta.ca (Andrew Simmonds) writes:
> I am sure this has been covered before....
> I am having trouble getting TIGHT bands with Northern blots from
> formaldehyde RNA gels... When I cut off the ladder from the gel and look
> at it sideways the bands are "tilted" i.e. ( / ) and I am sure this is a
> problem when I transfer the RNA to a membrane causing the wide bands I am
> seeing. I put 2.2M formaldehyde in the gel and use a standards MOPS
> Might anybody suggest a better way?
> Andrew Simmonds
I seem to remember someone round here having the same problem, due to
formaldehyde diffusing out of the gel. They got around it by leaving the
formaldehyde out, and simply denaturing the RNA with formaldehyde before
loading. It seemed to work. I use glyoxylation for Northerns, as it gets
away from those nasty |-( smelling gels.
You get the same effect with DNA gels when only the buffer but not the gel
contains ethidium bromide. The ethidium slowly diffuses into the gel from
above, intercalating with the DNA and (because EtBr is positively charged)
progressively altering the mobility of the DNA in a way that makes the bands
"fall over" and tilt in the way you describe. This would only be a problem if
you had ethidium in the gel, which seems unlikely.
NNNN NN Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-0750
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