Amont of DNA to use for transient Transfection?? - (Jun/10/2005 )

Hi everyone,
I have a few basic questions regarding transient transfection of a adherent cancer cell line.

1. What is the amount of plasmid DNA that one uses for say a 24 well plate?
2. I normaly use 0.5 ug of plasmid per well in 500 ul of media. If I increase the media amount to 1 ml do I have to increase the amount of plasmid too?
Some people tell me that the amount of DNA is dependent on the size of the well and not the amount of media in there. Is this the right concept?

any help will be appreciated.
regards,
p..

hi
amount of dna depends on cell number. As cell number is evaluated by confluency, and if your reproducible regarding confluency, i assume we can say that amount of dna is dependant of plate surface. I've successfully used 1 to 2µg of plasmid dna for transfecting 6well plate. As surface of a well in a 6well plate is 4times surface of a well of a 24well plate 0.5µg is ok.

For medium quantity i would say that 1ml is quite a lot for a well for a 24well plate. In my experiments, i use 1ml per well. So i would say that 500µl is the max. But you can try trnasfection with 1ml. In such case DNA quantity should be the same as in 50µl.

Fred

Hey Fred,
Thnaks a million for the prompt reply. Needed it ASAP.
I do normally use 500 ul but I was just wondering as I was teaching someone and did not want to give a wrong answer to that guy.

Thanks a lot once more
pri