Subtractive Hybridization Pitfalls?

James Graham 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.
>
> Regards,
> 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.

Jim
J. Graham PhD 
Biology Department 



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