I’M trying to perform cotransfection for 293T cells in 12-well cell culture plate with two plasmids (X and Y) (DNA Conc. for X is 1016 ng/ul and 570 ng/ul for Y). I would like to use different ratios of plasmids in order to get accurate effeciency yield and expression for proteins of interest. I would be extremely grateful if you could share some advices.
Dear Ghadeer Abdullah ,
There are a few things you should take into account. First of all, depending on our Plasmids (size) 1000 ng of X may not be the same mount (copy number) as 1000 ng of Y so you should check your plasmid maps to use the same copy numbers (there are calculators for that in the internet).
Another problem you might face is the use of different promotors (i.e. CMVie is by far stronger than, let's say PGK or EF1alpha) with in your construct, so that the amount of mRNA generated by the same copy number might be different. Also you might think about protein stability from your inserts that might also differ. So to solve now all problems at once I would like to suggest to do Western-blots with monoclonal antibodies (to solve the problem of signal amplification by the usage of polyclonal mutible binding).
You should start with i.e. a 6-well per condition and transfect 2 or 3 µg DNA (i.e. 1000 ng of a 5000 bp plasmid X and 1200 ng of a 6000 bp plasmid Y - since it is 1/5 bigger you schould use 1/5 more to reach the same copy number) by CaPO4 method.
Using the same copy number of plasmid. Than you might be able to transfect variations of your plasmids (i.e. 1:2, 2:1, ....). But if your Plasmids are using Promotors like the CMVie and you are using a high amount of DNA (i.e. 2000 ng/ 6-well), plasmid DNA amount might not be the limiting factor for your expression.
This is at least my first advice how I would approach the problem. Your question is a little bit limiting. Where are you really struggling?
Kind regards
Soenke
Thank you Nikhil Dev Narendradev . I actually will use Lipofectamine® 3000 reagent, Do you think I still needing to incubate DNA in ice for 30 minutes before transfection?
Thanks Sönke Weinert ! I had tried several times using 6-well, but the yield of protein was low.
Dear Ghadeer Abdullah ,
I assume that the inserts you would like to express are not tagged with a fluorescent protein. Have you checked your transfection efficiency with a GFP vector and subsequent fluorescent microscopy or FACS analysis. It may not be a problem of your plasmids, but with your transfection.
Nikhil Dev Narendradev, I appreciate your help for explaining several factors that could caused a low yield of my target.
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