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DAPI questions

RPSCOTT RSCOTT at IRRI.CGNET.COM
Mon Aug 23 03:18:47 EST 1993


> 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!

Scott

======================================
R. Scott
Division of Plant Pathology
International Rice Research Institute
POB 933, 1099 Manila, Philippines
e-mail: rscott at irri.cgnet.com
======================================





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