transfection with ligation mix? - (Feb/24/2008 )

Hi!
I have been struggling with cloning my gene into pcDNA3.1 vector in E.coli. It seems the gene is toxic for E.coli if it placed after CMV promoter. I've read CMV might works in E.coli as well. I am able to clone it in the opposite orientation but not in the right one.
So I am thinking about to transfect the ligation mix directly (or after a further linearization) into mammalian cells.
Does anyone have experience on that?! I am using lipofectamine and want to make stable transfectant.
thanks,
Attila

Maybe this will interest you: a proper designed PCR product with a promoter, a coding sequence and a terminator is functional in mammalian cells:

http://www.jbc.org/cgi/content/abstract/M110652200v1

well, you might not have adequate DNA for efficient transfection. but definitely there might be a possibility to get a few cells.

good luck !!!

Thanks for the advices!
Well that's an option, ligate the cDNA and make a PCR with primers spanning all the necessary elements and transfect with that.
Although I am a bit concerned about introducing mutations.
I'll let you know the results...

mutations ... would depend on the size of your transcript and what kind of proof reading polymerase you are using. If you kept down the number of cycle but conduct several PCR reaction you should have enough material.


hmmm... and if you are considering putting the cDNA into a plasmid... can't you make alot of plasmid and just cut out the fragment that you want and transform it?

EDIT: Ops forgot to reread the heading.

The only way this is going to work is if you are certain that you are cloning the insert into the vector in the proper orientation. You assume that the gene is expressing in e.coli and those that get the plasmid with the properly oriented insert are dying. There could be other reasons, such as secondary structure of the insert, which are inhibiting the ligation in the proper orientation. Before you transfect and select for a couple weeks you may want to try to re-digest the ligation with enzymes that will show orientation. You'll get a mix (hopefully) but as long as the bands of the correct size show up you should be able to get a stable cell line with the ligation. Otherwise you could try to do PCR screening of the ligation. Use a vector specific primer and an insert specific primer and see if you can detect the insert in the proper orientation. It just seems like a lot of work to try to make a stable cell line when you aren't certain that the ligation is producing the correct orientation.