Drug
Cells:
Materials:
2xSPP medium
200x fungizome
1000x penicillin/streptomycin
1 mg/ml CdCl2 (stock
solution is 1 mg/ml)
10 mg/ml Paromyicin
(for neo3 cassette)
96 well tissue culture plates
100 mls
of 2x Spp (+1x penicillin/strepomycin
and fungizome +1 mg/ml CdCl2)
The
next day, add 25 mls of 2x Spp
(+2x penicillin/streptomycin and fungizome) plus 1
mg/ml CdCl2 to each flask containing cells in 10 mM
Tris (final CdCl2 concentration is 0.5 ug/ml)
Incubate
at 30 degrees Celsius 5 to 7 hours with slow shaking.
Add
additional CdCl2 to 1 ug/ml (25 ul of 1 mg/ml stock)
Add
10 mg/ml paromomycin to 75 to 80 ug/ml
(we usually use 80 ug/ml)
Pour
cells into media tray
Plate
into four 96 well plates (125 ul/well).
Incubate
at 30 degrees Celsius at least 3 days before checking for transformants.
Optional: add 10mls of 1xSpp +1 ug/ml CdCl2+ 80 ug/ml paromomycin (+1x penicillin/strepomycin
and fungizome) back to each 50 ml flask and incubate
to recover any transformants remaining in medium not
plated.
Cole Lab:
Make 20 ug/mL stock solution.
In TRIS or NEFF dilute 1:100 for final conc of
0.2 ug/mL. Can be higher up to 0.5 ug/ mL
From Chalker:
Our
SPP and Neff media contain sequestrine. For inducing cells during conjugation, if i want to induce upon mixing (as for PDD1-CFP), I add CdCl2
to 0.05 ug/ml (this dose does not interfere with
pairing), and 3 to 4 hours later, I will boost it to a final concentrations of
0.1ug/ml. If I want to induce later in
conjugation, I will usually induce between 0.08 and 0.1 ug/ml. One can go a little higher, but then it can
have toxic effects.