Stable transfection of Schneider 2 cells
Materials:
plasmids (e. g. pMT, pUAST)
pCoHygro plasmid
transfection reagent (e. g. Fugene HD)
confluent S2 cells, should not be old
Schneiders medium (M3, serum free)
Schneiders medium (M3 with serum, Pen/Strep and hygromycin (0.3 mg/ml final conc.)
Procedure:
- Day 1: Seeding
seed cells with 1.5x106 /ml in a 25 cm2 bottle in 5 ml medium.
Use actively dividing cells, e. g. two days after splitting.
- Day 2: Transfection
transfection with
24 ug DNA (1,26 ug pCoHygro (1/19) + 22.74 ug plasmids)
60 ul Fugene HD (DNA : Fugene = 1:2.5)
ad 300 ul (final volume) with Schneiders medium without supplements
pipette first the Schneiders medium
then the DNA
last the Fugene reagent (invert the vial before pipetting)
mix by tipping with the finger (Note: Minimise the contact of undiluted FuGene HD with
plastic surfaces. Do not mix by pipetting up and down. Pipette directly
into the solution)
incubate for 30 min at room temperature (please see the Fugene manual)
add the transfection mix drop-wise to the bottle. Swirl the
flask to uniformly distribute the medium over the entire surface.
- Day 5: Selection
change to a medium with 300 ug/ml Hygromycin B
- Day 10ff: Selection
Change the selective medium every five days. After about six weeks there should be a
sufficient number of resistent cells. During this time only exchange the medium every 4 to
5 days. Do not split the cells. Move the bottle as little as possible. Take care
not to rinse off the cells. If resistent colonies do not form properly, the cells
can be incubated without exchange of the medium for up to two weeks.
Notes:
S2 cells have to be at a minimal density of 1x106 per ml in order to proliferate.
You may need to concentrate the surviving cells, putting them into a smaller container.
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