Protocol No. 4308 915.031 12/1999
CHO, Chinese hamster ovary,
Rat-DPP IV (in bidistilled H2
O), stable transfection
Eppendorf Hypoosmolar Electroporation Buffer (PH)
Alpha-Medium / 10% FCS
Eppendorf, 2 mm gap width, 400 l
RT (20-25 C)
Sabine Grams Institut fr Molekularbiologie
und Biochemie Arnimallee 22
D-14195 Berlin Phone +49 30 8445 1543 Fax +49 30 8445
- Harvest the cells in the exponential growth phase and centrifuge
them (for 5-10 minutes, 200 x g, at room
- Resuspend the cells in Alpha-Medium / 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 Hypoosmolar Electroporation Buffer. When doing
so, set the cell concentration to
1 x 106<
- Add and mix plasmid DNA (5-10 g/ml final concentration, in
- Transfer 400 l cell suspension into electroporation cuvettes
(2 mm gap width). The cell suspension must be free of
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 Alpha-Medium
/ 10% FCS, centrifuge it for
5-10 minutes, 200 x g, at room temperature. Remove supernatant.
- Resuspend the cells in 2 ml Alpha-Medium / 10% FCS, and cultivate
them in a 6 well microtiterplate.
Detection methods for transfection:
98% (measured 48 hours after transfection)
Transfection rate (stable):
50% based on the initial number of cells used for the
experiment (after 10 days of incubation
in selection medium)
Source:Page: All 1 2 Related biology technology :1
. Borrelia burgdorferi