Hi, I need to transfect a melanoma cell line with a plasmid but I’m not sure how much DNA (ug) is necessary for the transfection and which plate is better for this, 96, 24 or 6-well plate.
I accept any advice, protocol and suggestions
Thanks!
I transfect MDA breast cancer cells with Lipofectamine 3000 and transfect about 1200-1600ng of plasmid DNA. My plasmid expresses really well so hence the possible lower end concentration. Try to follow the manufacture's instructions. I have only transfected in 6 well plates and it works fine. Also I use half of what they recommend for P3000 and it works just fine (but test it first).
We routinely perform transfections in our lab and have used the 6 well as well as 24 well format. Although 96 well should also be easy to use. Id suggest you to try 6-well plate transfection with 1.5- 2.0 ug of your favorite plasmid. If you trying to standardize transfection in its will be ideal to include a well for GFP transfection as a positive control which can be visualized next day itself. Also make sure you use high quality plasmid extraction method (midi prep) as mini-prep extraction have endotoxins, which significantly reduce transfection efficiency.
Goodluck!
Fernando,
We do regular transfections using L-3000. It works nicely. You can not experience cytotoxicity like others(L-2000) and simple protocol.
1)Take recommended amount of Serum free antibiotic free medium in tubes(equally)
2)Add P-3000(2"x"'ul) and "x"'ug of plasmid to one tube. For second tube add "x"ul of L-3000 reagent & incubate both for 5 min.
3) Mix both solutions and incubate for 10-20 min and finally add to the cell culture plate.
No need to change medium(10%FBS DMEM/RPMI with Antibiotics) before and after since it works based on CRISPR-cas9 method untill unless required. Plate type, duplicate or triplicate and DNA concentrations("x"'ug) depends on your experiment as Dr. Joel Mentioned. All the best.
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