Protocol No. 4308 915.050 11/2001
721.221, EBV transfected B cell line
pcDNA3.1/V5-His-TOPO (in bidistilled H2
Eppendorf Hypoosmolar Electroporation Buffer (PH)
RPMI / 10% FCS (200 U/ml penicillin; 200 g/ml
streptomycin, 2 mM glutamine)
Eppendorf, 4 mm gap width, 800 l
RT (20-25 C)
Dr. Britta Eiz-Vesper Medizinische
Hochschule Hannover Institut fr Transfusionsmedizin
Feodor-Lynen-Str. 21 D-30625 Hannover
Phone +49 160-3680653 Fax +49 511-5322079 e-mail: firstname.lastname@example.org
Detection methods for transfection:
- Harvest the cells in the exponential growth phase and centrifuge
them (for 5-10 minutes, 300 x g, at room temperature).
- Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells
and centrifuge them (for 5-10 minutes, 300 x g, at room temperature).
Note: The overall incubation time in the Eppendorf Electroporation
Buffer must not exceed 30 minutes to guarantee a successful electroporation!
- Resuspend the cells in Hypoosmolar Electroporation Buffer. When doing
so, set the cell concentration to 2 x 107 cells/ml.
- Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled
- Transfer 800 l cell suspension into electroporation cuvettes
(4 mm gap width). The cell suspension must be free of air bubbles.
Time constant (T)
No. of pulses (n)
- After the pulse, allow the cell suspension to stand in the cuvette
for 5 minutes at room temperature.
- Carefully transfer the cell suspension with a pasteur pipette from
the cuvette into 5 ml RPMI / 10% FCS, and cultivate
them in a little culture flask.
Selection and limiting dilution assay for stable transfection after
40% based on the number of surviving cells.
Results were measured 48 hours after transfection.
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