Hi Rajesh,
I would suggest to use nucleofection if you have the possibility to access the equipment. We use Amaxa electroporation system (lonza), following provider's protocols reaching 30-50% efficiences. For primary embryonic hippocampal and cortical cultures.
If you don't have access to this type of setup, you can also try lipofectamine-based transfection. I used lipofectamine 2000, but you can check others.
For a 3,5 cm petri dish and 1.5ml final volume.
A: petri dish plus cells, add 1ml of Optimen (Neurobasal or N2 without serum can replace Optimen) leave in incubator for at least 1hr.
B: Mix 250 ul of Optimen plus your DNA (I try to use up to 5ug).
C: Mix 5-7 ul of lipofectamine2000 with 250 ul of Optimen. Leave at Room temperature for 10 minutes.
D: Mix B and C and leave 30 minutes at room temperature (better in an orbital shaker).
E: mix D and A and leave in incubator for 1-2 hs.
Finally replace the transfection media (without washing) with the neuronal media of choice.
Cheers!
Hi Rajesh,
I would suggest to use nucleofection if you have the possibility to access the equipment. We use Amaxa electroporation system (lonza), following provider's protocols reaching 30-50% efficiences. For primary embryonic hippocampal and cortical cultures.
If you don't have access to this type of setup, you can also try lipofectamine-based transfection. I used lipofectamine 2000, but you can check others.
For a 3,5 cm petri dish and 1.5ml final volume.
A: petri dish plus cells, add 1ml of Optimen (Neurobasal or N2 without serum can replace Optimen) leave in incubator for at least 1hr.
B: Mix 250 ul of Optimen plus your DNA (I try to use up to 5ug).
C: Mix 5-7 ul of lipofectamine2000 with 250 ul of Optimen. Leave at Room temperature for 10 minutes.
D: Mix B and C and leave 30 minutes at room temperature (better in an orbital shaker).
E: mix D and A and leave in incubator for 1-2 hs.
Finally replace the transfection media (without washing) with the neuronal media of choice.
Cheers!
Hello
Yes this good protocol , also see link here
Hope help you
Good Luck
http://www.jneurosci.org/content/30/18/6171.full.pdf+html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052554/
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