Cotransient Transfections DNA Requirements - (Mar/13/2012 )

I am trying to co-transiently transfect CHO cells with two different expression vectors. The protocol I am using requires 4 ug of DNA for a transfection. Should I use 2 ug of each expression vector or 4 ug of each expression vector?

Thanks!

Have you tested each expression vector in your system? If so, you should use the amounts of each that work.

Note that depending on your transfection system and the size of your assay, 4 ug is a huge amount of DNA. I routinely work in 6 well plates with 0.25-1 ug of DNA for most plasmids.

bob1 on Thu Mar 15 20:15:53 2012 said:

Thank you! So you are suggesting that I test each vector individually, and then to co-transfect with each optimized amount of DNA, regardless how much total DNA is suggested for each transfection?

The protocol I am using suggests using 4 ug/well for 6 well plates and .8 ug/well for 24 well plates.

If you want to keep the total to 4 ug, you should test each individually to see the maximal expression (note that more plasmid doesn't necessarily mean more expression), and work from there.

You can also use as much or as little plasmid as you like, if you need to use 8 ug, go for it, but beware of the potential for toxicity from the plasmids and/or the transfection reagent.

The amount of DNA to use depends on the transfection reagent to some exent; Fugene, one of the more effective reagents, says to use a maximum of 2 ug for a 6 well plate, whereas PEI recommends 3 ug.