svetlov at oncology.wisc.edu
Wed Apr 8 23:39:04 EST 1998
In article <01bd6302$b2a84ae0$d966c180 at kortej>, "John Korte"
<kortej at css.orst.edu> wrote:
> I have a question or two about the most effecient way to run a southern.
> How much DNA do you load & does it make any difference if the DNA is
> RNased? (If it makes any difference, in our lab we have a vacuum blotter
> and a roller oven.)
The most efficient way to find out about that is to check papers on DNA
fingerprinting from Sir Jeffreys in the 80's. I remember there was even a
fingerprinters' only leaflet circulated where people discussed different
tricks to make the blots look better (such as the best ratio of comb
tooth's width to length (1:10 - the magik numbers), thickness of buffer
overlay etc.). I can't remember all of that by heart so if you can find
some of the good olde paper (about pre-PCR fingerprinting that is) you are
gonna get a pretty good idea how to run a nicah lookin' gel. Removal of RNA
does improve the looks and separation, amount of DNA depends on the size of
the well and the level of digestion (DNA digested with 4-cutters can be
loaded in greater amount than the same DNA cut with 6- and more cutters),
run slowly - o/night in TAE, rather than in TBE if a good separation of
bands over a broad range of sizes is desired. Don't overlay it with more
than a couple of mm of buffer. I like roller ovens but find vaccum blotters
less consistent than capillary or electroblotters.
Vladimir Svetlov, Ph. D.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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