- Make a stock of gelatin media
by using 5 ml of whatever minimal media you want to use (for example,
you may want to use media that will induce your fusion protein) and
adding 25% of gelatin off the shelf. Heat this in a hot
water bath until the gelatin melts. Now you can store this to
use (make sure you try to keep it sterile, because things will grow
in there).
- When you film, grow cells
to log phase (0.5-0.8 OD) and then spin down about a ml in a 1.7 ml
epp tube. Resuspend in about a ml of H2O and then
spin down again. Then resuspend in about 50 ul of H2O
to concentrate them (you may want to very this amount depending on
how thick the culture is).
- Make a slide by using the
Beach/Maddox method. This is where you take two clean slides
and offset them a little so that they are face to face. Clamp
one end, on the very end, with a large binder clip (the black ones
we use for papers that are too big for staples), make sure it's
the really big kind. Heat your gelatin/media in a water bath
until it is fluid, then pipet about 50 ul in between the slides.
This is sort of tricky at first, and why you offset the slides a little,
what you do is pry them apart using the clamped place as a hinge so
that you can fit your pipeter in there. and then let the slides
come together, smashing the fluid. Clamp the other side with
another clip. Briefly pass ONE SIDE of the clamped slides over
a flame, this will melt the gelatin a little extra and will make it
spread better. Let the slide cool down for about 10 to 15 minutes
before you take the clips off. After removing the clips, carefully
pry the slides apart, a razor blade sometimes helps. This takes
practice.
Put 3-5 ul of cells on the gelatin and then cover with an 18x18 cs.
Press the cs down a bit and then seal with valap.
Questions? Email Paul at pmaddox@email.unc.edu
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