Protocol No. 4308 915.039 12/1999
Embryonic stem cells (ES cells), mouse, R1. Passage
Linearized targeting vector (12 kb) with neoR
Eppendorf Hypoosmolar Electroporation Buffer (PH)
DMEM / 15% FCS, 2 mM glutamine, 1 mM pyruvate, 1 x MEM,
1 x nucleoside mix, 1 x Pen/Strep, 1000 U LIF
Eppendorf, 4 mm gap width, 800 l
RT (20-25 C)
Dr. Michael Bsl ZMNH
Transgene Technologie Martinistr. 42 D-20246 Hamburg
Phone +49 40 42803 6663 Fax +49 40 42803 6659 e-mail:
- Harvest the cells 36-40 hours after the last passage, determine the
number of cells and centrifuge the cells (5 minutes, 200 x g, at room
- Wash the cells in Hypoosmolar Electroporation Buffer: Resuspend the
cells in 10 ml Hypoosmolar Buffer and collect them by centrifugation
(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 at a concentration of 1 x 107 cells/ml
in Hypoosmolar Electroporation Buffer.
- Mix 750 l cell suspension with 50 l DNA solution (1 g/l in Isoosmolar
- Transfer 800 l cell/DNA solution 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, let the cells shortly recover for 5 minutes at room
temperature and then carefully transfer the cells to 10 ml ES-medium.
- Plate the cells onto 100 mm culture dishes prepared with inactivated
mouse embryonic fibroblasts feeder layers at a density of 1-1.5 x 106
cells per plate.
- The next day change medium and start selection 24 hours after the
electroporation with 250 g/ml medium of G418. Change selection medium
- 7-8 days after the electroporation pick the surviving individual ES-cell
colonies under a stereo microscope, culture and expand them individually.
Take only colonies with sharp borders and proper morphology.
- After expansion freeze down at least one aliquot of each clone and
use another for DNA preparation to identify correctly transfected clones
originating from homologous recombination by PCR or Southern Blot analysis.
Detection methods for transfection:
Selection with G418 to identify G418 resistant, stably transfected colonies
carrying the neoR
marker of the linearized targeting vector.
Source:Page: All 1 2 3 Related biology technology :1
. Primary cells, rat heart endothel2
. Primary cells, rat heart muscle