reporter vector cotransfection - (May/19/2007 )

hi everyone i need your help!
i'm going to do transfections in SHSY5Y cells with my plasmid(which contains the transcriptional factor) a second plasmid with tha response ellement and the luciferase gene and a third with β-gal for normalization of the results. Does anyone knows about the amount ratio of the three plasmids? is it acceptable to put equal amounts?
Or do I have to increase the amount of the reporter gene(luc) for best results. I'm going to compare mutated trans.factors.
thanks a lot
maria

-manou-

QUOTE (manou @ May 19 2007, 04:27 AM)

I would compare the effects based on equal amounts of DNA of each plasmid and other with equal molar amounts based on their size. Choose from one of these, optimize transfections and do the experiments.

-scolix-

thanks for the answer. I wonder if there is a reference or a common strategy for the reporter plasmids since is a very usual assay.

-manou-

There is no fixed formular. You need to decide based on the assay sensitivity, the strength of the promoter and the activity of the transcription factor. b-gal assay is less sensitive if you use optical measurement with ONPG, so you may want to use more b-Gal vector. Luc reporter typically are more easy to measure, therefore you can use less. I would suggest you to test it with a ratio of 1:1:2 for transcriptional factor construct, luc reporter and b-Gal normalizing vector, and do adjustment/modification on the ratio from there.

-genehunter-1-

i focus on molarities of plasmid, as if one is 10kpb and the other is 5kpb, equal mass don't mean equal molarity.

-fred_33-