RNA electrophoresis

Joel A. Kreps jkreps at SCRIPPS.EDU
Tue Nov 4 12:05:54 EST 1997


Dear Han and others,

I routinely run formaldehyde gels (0.55M formaldehyde) and get excellent
staining and probe-able northerns.  I follow essentially the same
methods as described by Han (i.e., 10XMOPS, agarose gel + formald.),
however, I add EB to my RNA samples not the gel.  I have ~0.1 ug/ul in
my loading buffer (formamide, formald, 10x MOPS, EB) and add 10 to 15 ul
per RNA sample.  I mix the loading buffer with the RNA, heat at ~75oC
for 15 min. quick chill on ice, add loading dye (xylene
cyanol/bromophenol blue, ficoll, edta), spin the samples and load.  I
get beautiful staining, essentially no background and my gels look like
regular DNA/TBE gels in terms of staining.  There is extra EB in each
sample, and the EB runs in the opposite direction (neg to pos), so I do
get big splotches above the wells (a problem when running double lanes
on one gel).  I soak the gel for 30 min. in 10X SSC and transfer onto
nylon or nylon reinforced membrane with 10XSSC and have done many tens
of hybridizations and strippings with no problems.

Joel A. Kreps
The Scripps Research Institute
La Jolla CA



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