two questions

David Alexander dalex at NEXUS.MICROIMM.MCGILL.CA
Thu Aug 14 10:06:55 EST 1997


Two questions for the bio netters!

ONE
I am interested in conducting pulse-chase experiments to
monitor DNA synthesis/replication in a prokaryotic system (Caulobacter)
The references I have found are too vague to be of much use.
Can anyone suggest papers/protocals that describe 
prokaryotic pulse-chase experiments?


TWO
I need to linearize a large and unruly plasmid (almost 30 kb)
The obvious method is with a restriction enzyme.
However, the enzymes I have used either refuse to cut,
or chop the plasmid into many fragments.
I have sequence data for portions of the plasmid, but not all of it,
so restriction site analysis has been of limited use. 

Are there commercially availble enzymes that might be of use?
(eg something that cuts non-specifically and infrequently)
Would it be possible to use DNase under less than ideal conditions?
What about chemical cleavage methods?

Any suggestions would be much appreciated.


David.

dalex at microimm.mcgill.ca  



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