Multiporator
Transfection Protocol
Protocol No. 4308 915.024 11/2001
Cell line
H73C11, mouse-human, heteromyeloma (suspension cell
line)
Transfection with
Plasmid pEGFP-N1 (in bidistilled H
2O)
Electroporation buffer
Eppendorf Electroporation Buffer with 150 mOsmol/kg
Culture medium
RPMI 1640 / 10% FCS
Cuvette
Eppendorf, 2 mm gap width, 400
Temperature
RT (20-25 C)
Reference
Prof. Ulrich Zimmermann Lehrstuhl
fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508
Fax +49 931 888 4509
e-mail: zimmerma@biozentrum.uni-wuerzburg.de
- 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).
Remove supernatant.
Note: The overall incubation time in the Eppendorf Electroporation
Buffer must not exceed 30 minutes to guarantee a successful electroporation!
- Resuspend the cells in 150 mOsmol/kg Electroporation Buf
fer (= 68%
Eppendorf Hypoosmolar Electroporation Buffer + 32% Eppendorf Isoosmolar
Electroporation buffer). When doing so, set the cell concentration to
1 x 106 cells/ml.
- Add and mix plasmid DNA (10-20 g/ml final concentration, in bidistilled
H2O).
- Transfer 400 l cell suspension into electroporation cuvettes (2 mm
gap width). The cell suspension must be free of air bubbles.
- Electroporation:
Mode
Eukaryotes
Voltage (V)
160 V
Time constant (T)
40 s
No. of pulses (n)
1
- 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.
Detection methods for transfection:
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.
Result:
Survival rate:
250%
Transfection rate:
20% based on the initial number of cells used for the experiment
Results were measured 48 hours after transfection.
'"/>Source:
Page: All 1 2 3 Related biology technology :1.
Primary cells, rat heart endothel2.
Primary cells, rat heart muscle