Single cell sorting (long, w/ no trivial musical references)

Steve G. Hilliard (steve@habanero.cb.uga.edu)
Mon, 3 Mar 1997 15:42:26 -0500 (EST)

Hi folks,

A few weeks (months?!) ago I asked about sorting single sperm for PCR,
and recieved quite a few encouraging responses. Th interested party has
obtained the dye (we're using Ho348) and tested their PCR after staining,
and they're quite satisfied and ready to sort.

Now the ball is in my court....
We have a 753 with Cicero. I dug out our auto-clone unit, got the 753
sorting beautifully with the normal sort drawer, and then installed
the auto-clone. Now I'm stuck! The next step is calibration and
testing, to adjust the instrument so that the sorted test stream
(actually the "unsorted" stream) goes through a hole in a plate in the
sort drawer. The multiwell plate is underneath this hole, and any droplet
that is going to land in the well must go through this hole. Problem is, my
stream won't go through the hole! The waste (what I would call the
center stream and the left stream) is deflected to the left and lands in
the waste catcher, but the stream I want (the right stream, which should
be falling straight down) is too far to the right. The manual
indicates that I should adjust the right-left and front-back knobs on the
autoclone until the stream goes through the hole, but the
right-left knob has no noticeable effect. Does anyone know what it
does? It looks like a potentiometer, and I'm speculating that it
modulates some bias in the plate charges to shift all streams
right or left. I thought about yanking it and substituting one w/ a
larger range of resistances, but there should be better way. I tried
decreasing deflection gain, and that does move it a bit to the left, but
not enough, and I'm limited by the fact that at too low a setting the
waste streams start splattering off the edge of the waste catcher.

Does anyone have any ideas? I won't be able to work w/ the instrument
again for another week, but we really need to get this working.
Suspecting that deflection in one direction may be higher than the
other, I'm going to try switching the plate polarity and cable
setups so that right is left. Does anyone have any experience w/ this
beast, or know of any other tricks?

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Steve G. Hilliard Join the fight--help
Cell Analysis Facility stamp out smartass
University of Georgia sig files!
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