Electroporation of nec: Difference between revisions
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(Created page with "== Electroporation == # Incubate NSC 60-90min with 1mM DPBS/EDTA # Prepare 1.5ml tubes with DNA mix (max. 3µg/µl, 0.75-1µg/µl per plasmid). Max. 3µl DNA mixture # Prepare...") |
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== | == Procedure == | ||
# Incubate NSC 60-90min with 1mM DPBS/EDTA | # Incubate NSC 60-90min with 1mM DPBS/EDTA | ||
# Prepare 1.5ml tubes with DNA mix (max. 3µg/µl, 0.75-1µg/µl per plasmid). Max. 3µl DNA mixture | # Prepare 1.5ml tubes with DNA mix (max. 3µg/µl, 0.75-1µg/µl per plasmid). Max. 3µl DNA mixture | ||
Revision as of 09:07, 25 March 2015
Procedure
- Incubate NSC 60-90min with 1mM DPBS/EDTA
- Prepare 1.5ml tubes with DNA mix (max. 3µg/µl, 0.75-1µg/µl per plasmid). Max. 3µl DNA mixture
- Prepare 12well plate with gelatine
- Prepare Culture Media
- Centrifuge cells for 3min at 700g
- Resuspend one flask (75cm2) with cells after centrifugation in 160µl Steinberg solution
- Add 12µl each of the cell suspension to every DNA mix
- Electroporation conditions are: 500V, 50ms, 5 pulses
- Add cells to the plate and check after 1 day
Culture media
| Name | Quantity | Remarks |
|---|---|---|
| Glutamax | DMEM:F12 | |
| Glutamin | 10µl/ml | |
| Pen/Strep | 10µl/ml | |
| B27 (50x) | 20µl/ml | |
| FGF2 (20µg/ml) | 20ng/ml | |
| Shh (Curis) | 100ng/ml | For lower passages only |