whole-cell hybridization, HELP!

Andrew St. Jean andrew at bio01.bio.uottawa.ca
Wed Aug 28 09:34:18 EST 1996


Dear All,

   I am looking to do some whole-cell hybridization with fluorescent
probes, something I have not done before and I have a couple of
questions.

   First, I am wondering about sensitivity problems. I want to detect
the presence of a plasmid which is low copy number in the cell, probably
around 10. This plasmid is big, 442 kbp, and we have the whole thing
cloned into cosmids. I anticipate that I will need to make multiple
labeled probes that hybridize along most of the length of the plasmid
but I am unsure whether even this will be enough. Also, everything I've
read talks about hybridizing to RNA. Is it possible to do DNA-DNA
hybridization with fluorescent probes?

Second, the organism I am using is a halobacterium, specifically
Haloferax volcanii. This Archaeon requires at least 1.5 M NaCl in its
medium otherwise it falls apart. Does anyone know is whole-cell
hybridizations have been done on extremely halophilic prokaryotes? I
expect the fixing and hybridization solutions will need to be modified
accordingly.

   Please send replies to Andrew St. Jean at:
		andrew at bio01.bio.uottawa.ca

   Any help will be greatly appreciated.

Andrew St. Jean
University of Ottawa



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