I want to do a stable luciferase transfection of murine PDAC (PANC02) and colorectal (CT26) cell lines. Please help me find where to obtain the protocol and reagents.
Thank you.
Sincerely,
Lakshmi.
Hi Lakshmi,
Many of the protocol details that you asked for would be specific to your cells. You will need to identify a transfection reagent and conditions for your specific cell type and go from there. Alternatively, you could also consult publications to get an idea. Fugene HD or Lipofectamine are pretty popular and a likely a good place to start. You would optimize the transfection conditions according to the manufacturer's protocol. In general, your luciferase construct would need to contain a mammalian selection marker (e.g. puromycin), and most of the time you would start selecting for your stable clones 48h after transfection. You would also need to optimize the dose of the antibiotic that you are using to select stable clones for your specific cell type(you could consult publications as a starting point).
Hello Dr. Akansha Singh,
Thank you very much for your suggestion.
I found the lipofectamine 3000 transfection protocol on Invitrogen. I also found many pGL3 luciferase plasmids that are commercially available (on addgene and Promega), are plasmids is suitable for lipofectamine-mediated transfection? or do I need a lentiviral vector for plasmid transfection? How do I select the plasmid suitable for my cell lines, what are the criteria I should consider?
K15/pGL3
(Plasmid #44264)
Hi Lakshmi,
As long as the vector you are using has a mammalian selection marker, you should be able to select. You do not need a lentiviral construct unless you are planning on infecting. You can use it if you want, but it is not necessary. The plasmid itself is not going to be "unsuitable" for your cells, but rather transfection protocol needs to be optimized for different cells (so it's really more about the reagent). Some sequences can be toxic to certain cells, but this is rare in the case of luciferase constructs (it comes up more if you are over-expressing something). If a plasmid is too large it will transfect less efficiently. Beyond this, you should be able to follow general protocols and optimize your experiments.
Try linearizing the plasmid before transfection. It would be efficient. Try using lipofectamine and also use the selectable marker after 4-5 days to check the cloning.
- Badges
- Science method