spectrum for EtBr-DNA

Stephen Kelley kelley at vet.vet.purdue.edu
Tue Jul 9 10:52:39 EST 1991


In article <1991Jul9.145323.19439 at menudo.uh.edu> bchs1b at JANE.UH.EDU writes:
>I want to measure EtBr-DNA complexes quantitatively using a fluorimeter.
>Does anyone know the appropriate wavelengths for excitation and emission?
>I know the main excitation wavelength is around 300nm, but can you also
>excite at a higher non-UV wavelength (even if not as efficiently)???
>

Shapiro, _Practical Flow Cytometry_, says there is an excitation peak
with half maximum points at about 460 and 570, and one tenth maximum
points at about 425 and 580.  Emission half maximum points are about
580 and 650 and tenth maximum at about 550 and 730.  Shapiro's presentation
is not quantitative, so I don't know the relative efficiency of the 320
excitation compared to the 510.

We usually use propidium iodide for DNA quantitation.  Spectrum is 
pretty similar, and we typically excite with a 488 argon laser line, and 
collect with 550 long pass filters.  488 isn't ideal, but it works
plenty well enough.

If you've got a scanning fluorimeter try scanning excitation between
400 and 600 while collecting around 630-650, then scanning emission
with the excitation peak.

For what it's worth, propidium will give you a slightly higher emission
wavelength, which may or may not help you in your application.

Hope this helps,

Steve Kelley            kelley at flowcyt.cyto.purdue.edu

Purdue University Cytometry Laboratories        (317) 494-8638  -- voice
B050 Hansen LSRB, Purdue University             (317) 494-0517  -- fax
West Lafayette, Indiana, 47907



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