Reagents, supplies, and
- cES media + antibiotic(s) (warm to 37ºC)
- Geneticin (G-418) at 200 µg/ml for vector containing the
neo cassette (used
for positive/single selection
- Hygromycin B at 200 µg/ml for hygro cassette
- Puromycin at 1.5µg/ml for pu-Δtk cassette
- Gancyclovir at 2 µM for vector containing the HSVtk cassette
selection (both positive and negative selection): Select
with G-418 or hygromycin B (at 200 µg/ml) + gancyclovir at 2 µM for
vector containing both neo or hygro and the HSVtk cassette.
Begin selection 24 or 48 hr after electroporation by feeding each
150 mm dish with 25 ml cES + G-418 (or hygromycin B or
Feed daily until all non-targeted colonies have died and surviving
colonies may be picked, usually 6-8 days.
- Begin selection 24 hr after electroporation by feeding each 150
mm dish with 25 ml cES + G-418 (or hygromycin B). Feed with single
selection media for 2 days.
- Begin second selection 72 hr after electroporation by feeding
all but one 150 mm dish with cES + G-418 (or hygromycin B) +
ganciclovir. Leave one dish with single selection media to use as a
control for double selection efficiency. There should be
considerable difference in the number of surviving colonies between
the single and double selection.
- Feed daily until all non-targeted colonies have died and
surviving colonies may be picked, usually 7-10 days.
Note: Vectors containing
DT (diphtheria toxin) as a negative selector, no additional
component is added to the media.
Written by Susan Hipkens