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I can answer your sperm sorting question, I think (!). Some years ago now,
we did this very thing. Initially we were sorting on scatter alone but
having indifferent results. Then we decided that putting a DNA stain in
would help identify the sperm as opposed to debris, tails etc.
Hoechst 33342 was the stain of choice so you are right there. 10ug/ml for
30 mins as I recall. For the sorting I gated around the haploid peak and
also used a scatter gate (using log amp for both forward and right angle
scatter).
The Hoechst did not appear to interfere with subsequent PCR and sorting
efficiency was good, but there were problems with sperm lysis as
they are tough little fellows. More details can be found in :
C.Williams et al Human Molecular Genetics 1993 4, 445-448
Hope that this is of some help
Derek
Derek Davies
FACS Laboratory,
Imperial Cancer Research Fund,
London, UK
http://www.icnet.uk/axp/facs/davies/index.html
On Mon, 13 Jan 1997, Steve G. Hilliard wrote:
>
> Hi folks,
>
> I have a lab here that wants to sort sperm for PCR, ie. they're not
> interested in X-Y determination, just sorting single sperm into
> 96-well plates for amplification. I was about to suggest staining
> with Hoechst -342 (we have UV) but does anyone have a feel for (a)
> the overall feasibility of this idea, and (2) whether Hoechst or
> any other DNA stain will interfere with PCR?
>
> While I'm in Pine, does anyone have any tipes for sorting chromosomes
> on a 753? How bout when n is greater than 35?
>
> Looks like it's gonna be an interesting year....thanks for any leads you
> can provide.
>
> Steve
>
> -------------------------------------------------------------
> Steve G. Hilliard This space available,
> Cell Analysis Facility email me for rates!
> University of Georgia
> -------------------------------------------------------------
>
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
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