nishir at ohsu.edu
nishir at ohsu.edu
Tue Aug 24 12:14:57 EST 1993
In article <01H21PB6K74W0001B3 at irri.cgnet.com> RSCOTT at IRRI.CGNET.COM (RPSCOTT)
>> From: IN%"<@CGNET.COM:gstuart at unixg.ubc.ca>" "gstuart" 21-AUG-1993 06:38
>> Hello! I need some technical information about the DNA-binding
>> fluorescent dye, DAPI. I am forced to use it in place of ethidium bromide
>> with our microscope system. Specifically, I would like any information
>> regarding preparation of stock solutions, working concentrations,
>> excitation and emission wavelengths, photo-stability, etc.etc. Any general
>> information would also be appreciated. Thanks in advance, Greg.
>Here are some info. on DAPI:
>o working stock for DNA staining [NAR (1990) 18(24):7461-7462]: 0.2 ug/ml]
>o excitation maximum: 344 nm
>o emission maximum: ca. 466 nm
>o preferred binding site: AT-rich sequence with mininum binding req't of
> at least 3 consecutive AT-pairs; long stretches rich in GC-pairs are
> therefore difficult to visualize with DAPI; with high GC content samples
> ethidium staining with or without DAPI will be better.
>You may also try using the fluorochrome Hoechst 33258 (bisBenzimide dye):
>o storage: 1 mg/ml [10 ul of this stock is mixed into 100 ml TNE solution
> prior to use; this is after the fluorometric estimation of DNA conc]
> TNE: 10 mM TrisHCl, pH 7.4; 1 mM EDTA; and, 0.1 M NaCl
>o excitation max: 356 nm (free dye); 365 nm (complex with DNA)
>o emission max: 492 nm (free dye); 458 nm (complex with DNA)
>o non-intercalating but has preference for AT-rich domains binding twice as
> much on dsDNA than on ssDNA
>Good luck, Greg!
>Division of Plant Pathology
>International Rice Research Institute
>POB 933, 1099 Manila, Philippines
>e-mail: rscott at irri.cgnet.com
In addition to the above info.... we have used both Hoechst and DAPI for
labelling nuclei when we do immunofluorescence. They are both very bright and
do not quench rapidly (my impression is that they quench at a slightly slower
rate than rhodamine). We have a Zeiss microscope and use a "DAPI" filter.
Both dyes are very forgiving and can be seen quite readily. They are so bright
that if you do immunoperoxidase, you can adjust the UV diaphragm open so that
you can see both nuclei and brown precipitate with bright field optics just
fine. Makes a nice color slide!
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