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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