Dominik Haudenschild, from
Koshi Kishimoto, Ver.10/22/04
1. Discard medium
2. Wash twice with PBS
3. Add 0.05% Trypsin/EDTA
4. Add
Medium (with serum) up to 5ml
This medium will be discarded at step 8. The use of PBS instead of medium
resulted in precipitation of plasmid DNA. ( I donÕt know why)
5. Pipette up and down to lift off cells
6. Transfer into 15ml tube
7. Cell count
8. Spin 1000rpm 5min
9. Add electroporation buffer (*) up to 5x10 6/ml. Dissolve the cell pellet by pipetting
10. Add plasmid at the final concentration of 25µg/ml
11. Incubate
at room temperature for 30 min
Shake several times to dissolve the precipitate
12. Transfer
into cuvette by 1ml disposable pipette
2mm and 4mm gap cuvette is equivalent to 400µl, 600µl respectively. If you need more cells than 3x10 6
(600µl), electroporate twice. (If 1ml, 500µl twice)
13. Electroporation
475V, 1ms, 4times, 1Hz for 4mm gap.
Cuvette interval is so narrow that disposable plastic pipette cannot
reach to the bottom. Glass pipette
with electric pipet-aid is useful to transfer after electroporation. Wash cuvette with PBS after
electroporation.
14. Transfer
15ml tube
Each tube has 5x10 6 cells x0.7 survival rate = approx. 3.5x10 6 cells/ pellet
15. Spin 1000rpm 5min
16. Discard electroporation medium, Add culture medium gently not to disturb the pellet
*Electroporation Buffer
75% cytosalts
120 mM KCI, 0.15 mM CaC1 2 , 10 m M K2HP04 , pH 7.6, 6.5mM
MgCl2
Ph was set by KOH
25%
DMEM (originally Opti-MEM I)