Dear Ann,
Regarding your second question ("2. When doing FRET analysis should the
laser with the lower wavelength always be in position 1?) my answer is that
it does not matter. Actually we are using a single laser in all line mode
with a prism and the first beam is the 514 nm line. This is what we use to
trigger data collection. The 488 nm line is below the 514 nm line, so this
line provides the delayed signal.
The important thing is that your filters should be good enough, they should
be blocked at the laser lines.
If one member of the donor-acceptor dye pair bleaches too fast, then you
might think about how to choose the order of the laser lines. But even
that case the illumination time is so short, the bleaching should be
negligible. In addition, all the samples (Donor only, Acceptor only,
donor+acceptor labeled and non-labeled, autofluorescence) should be
measured under the same conditions, so you will be able to perform all the
corrections necessary for calculating energy transfer efficiency.
Regards
Janos
At 04:14 PM 7/7/99 +0000, you wrote:
>
>Hallo Flowbies, (I hope that's not a rude word)
>
>2 Questions:FACS Vantage
>
>1. When sorting with dual laser does one calculate the drop delay from the
>2nd laser spot or from the first?. I've been using the 2nd and sorting
>seems to be o.k (haven't checked recovery) but someone else I know
>calculates from the 1st.
>
>2. When doing FRET analysis should the laser with the lower wavelength
>always be in position 1?
>
>Haven't been able to find anything regarding the above anywhere.
>
>
>slainte
>
>Ann
>EMBL
>Heidelberg
>
>
>
Janos Szollosi
Department of Biophysics and Cell Biology
University Medical School of Debrecen
Nagyerdei krt. 98, P.O.Box 39,
H-4012 Debrecen,
HUNGARY
Fax/Phone: (36) (52) 412-623
e-mail: szollo@jaguar.dote.hu
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