Electroporation of nec

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Revision as of 16:47, 29 November 2012 by Serghein (talk | contribs) (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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Electroporation

  1. Incubate NSC 60-90min with 1mM DPBS/EDTA
  2. Prepare 1.5ml tubes with DNA mix (max. 3µg/µl, 0.75-1µg/µl per plasmid). Max. 3µl DNA mixture
  3. Prepare 12well plate with gelatine
  4. Prepare Culture Media
  5. Centrifuge cells for 3min at 700g
  6. Resuspend one flask (75cm2) with cells after centrifugation in 160µl Steinberg solution
  7. Add 12µl each of the cell suspension to every DNA mix
  8. Electroporation conditions are: 500V, 50ms, 5 pulses
  9. 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
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