Subtractive Hybridization Pitfalls?
graham at biodec.wustl.edu
Wed Apr 24 11:10:28 EST 1996
> Look up some of the magnetic subtractive methods. Human Genome Sciences
> uses these methods and they have already reported the completed sequence
> of one bacterial species.
> Bill Alexander
I have been struggling with "magnetic beads" (Promega and Dynal) techniques.
The binding of these has been recently revised by Dynal downward some
90%, making them impractical for dealing with 20-40ug samples (eg. $120
tube for an single subtraction). I also find that Promega and Dynal beads
elute contaminants in formamide hybridization solutions (white ethanol
precipitate), and that large (9KB) DNA biotin-linked to beads shears off to
a great extent, or the Promega beads also shed a white precipitate
at 60C in 1X SSC without formamide as well.
The "St. John" method (NAR 16 (2) p. 10937, 1988) of seperating
driver-target -streptavidin with a simple phenol:chloroform extraction
(MUCH cheaper, apparenty as effective as hydroxyapatite methods) would
seem much better, unless I'm missing something. :)
Any comments on subtraction techniques most appreciated.
J. Graham PhD
More information about the Methods