I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen transfection system following the article "An electroporation protocol for efficient DNA transfection in PC12 cells" (Covello G, 2014, Cytotechnology). Result similar to Lipofectamin 2000.
Are there people using PC12 cells (CRL-1721, non-adherent and NGF responsive) who might have experience in transfection and who could share some knowledge?
Thank you very much
I was very satisfied with the transfection reagent Mirus TransIT-X2.
Try to get a sample
Hello,
I never worked with this cell type but I have a strong experience in transfection. You should also try lipofectamine 3000. The fold protein expression might be increase by two folds compared to lipofectamine 2000 (based on Thermofisher data).
Some advises to increase transfection rate:
- Increase the DNA/lipofectamine amount. For example I used 1 ug of plasmid per well (6-wells plate) with 3 ul of lipofectamine. I also performed 8 plasmids transfection (thus 8 ug of DNA with 24 ul of lipofectamine).
- When you prepare your transfection mix (solution A = DNA + optiMEM and solution B= Lipofectamine + optiMEM --> Add A in B, drop by drop, no vortex) and incubate 30 min at room temperature.
- Instead of growth medium, you should put optiMEM on your cell culture and then add your transfection mix. Incubate during 6 hours and then replace optiMEM/transfection mix by growth medium.
Good luck
thank you for your response.
When you have increased volumes, is it with the lipofactamine 2000 or 3000 kit?
Thank you
Lipofectamine 2000.
However I assume that you can use a similar strategy with Lipofectamine 3000, I never tried.
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