Kohara mapping membrane
kertesz at micro.biol.ethz.ch
kertesz at micro.biol.ethz.ch
Wed Sep 14 16:45:17 EST 1994
Hi netters!
We have partial sequences for a series of E. coli genes which we would now
like to map and clone. The easiest way to map these genes on
the E. coli chromosome seems (seemed?) to be to buy the Kohara phage bank
immobilized on membranes from Takara Shuzo (the "Gene Mapping membrane"), and
then simply hybridize with the same oligos that we had used for sequencing.
However, the guff that comes with the membranes doesn't give the tiniest hint
about how much DNA is actually on the membranes, and (you guessed it) we can't
see any signal at all using their protocols (correcting for the lower melting
point of the oligo, of course). All the literature references I've found
seem to use much larger probes, which is also a bit discouraging, and I
can imagine that if there is too little DNA on the membrane, the sensitivity
will simply be too low to see anything with, say, a 30-mer. Has anyone out
there probed these membranes with oligos and got useful results? And how many
times can one re-use them? The suppliers won't commit themselves, not
surprisingly, though they do supply a stripping protocol!
Thanks
Michael Kertesz
kertesz at micro.biol.ethz.ch
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