Looking for a simple, accurate nuclease assay

karlster karlster at excite.com
Wed Sep 8 11:17:21 EST 1999

One possible alternative which came to mind is using fluorometry. The
basic principle is a defined quantity of DNA template (specified by user)
is incubated with nuclease and, after an appropriate incubation period,
terminated (eg. by addition of EDTA). The terminated mixture is then
diluted with a Tris-EtBr solution and fluorescence level of the remaining
DNA-EtBr complex is measured. Theoretically with increasing enzyme level
or increasing time of incubation there should be concommitant decrease in
fluorescence due to loss of target DNA complexing with EtBr. If this
sounds interesting to you, Frank, just drop me an email and I'll try and
get you the specifics as far as filters, machine, references (aside - I
use fluorometry to do quick quantitations of miniprep DNAs but do not
myself perform nuclease assays as described above).



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