Protocol No. 4308 915.020 11/1999
Jurkat, T-lymphocyte, human leukemia (suspension cell
Plasmid pEGFP-N1 (in bidistilled H2
Eppendorf Hypoosmolar Electroporation Buffer (PH)
RPMI 1640 / 10% FCS
Eppendorf, 2 mm gap width, 400 l
RT (20-25 C)
Prof. Ulrich Zimmermann Lehrstuhl
fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508
Fax +49 931 888 4509
Detection methods for transfection:
- Harvest the cells in the exponential growth phase and centrifuge
them (for 5-10 minutes, 200 x g, at room temperature).
- Resuspend the cells in RPMI 1640 / 0.5% FCS, determine the number
of cells and centrifuge them (for 5-10 minutes, 200 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 Bu
ffer. When doing
so, set the cell concentration to 1 x 106 cells/ml.
- Add and mix plasmid DNA (5-20 g/ml final concentration, in
- Transfer 400 l cell suspension into electroporation cuvettes
(2 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 5-10 minutes at room temperature.
- Carefully transfer the cell suspension from the cuvette to 5 ml RPMI
1640 / 10% FCS, and cultivate it in a 60 mm culture dish.
Note: After pulsing, the cells should be incubated for 2-3 h at 37 C
before any centrifugation is performed, to ensure
resealing of the membrane.
The expression of the plasmid pEGFP-N1 can be detected clearly after
24-48 hours with the aid of FACS analysis or
under a fluorescence microscope.
67% based on the number of surviving cells
57% based on the initial number of cells used for the experiment
Results were measured
24 hours after transfection.
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