Difference between revisions of "Electroporation of nec"
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− | == | + | == Material == |
− | + | Culture media: | |
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{| class="wikitable" | {| class="wikitable" | ||
!style="width: 300px" | Name | !style="width: 300px" | Name | ||
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|For lower passages only | |For lower passages only | ||
|} | |} | ||
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+ | == 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 |
Revision as of 09:21, 25 March 2015
Material
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 |
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