NMDA Transfection in 293 Cells - (Jul/25/2011 )

Hello all,

I'm attempting to optimize my transfection conditions for NMDA receptors in HEK 293T cells. Currently I am transfecting with 9ug of N1, 9ug of N2, and 2ug of eGFP (for visualization). In the past I had great success with this, much so that I actually lowered my transfection volume down to 2ug for N1 and N2 subunits and still got robust expression. In the past month or two, my transfection efficiency has dropped down significantly and certain N2 subtypes are not expressing at all. Any suggestions on how to improve this? I feel that I need to up the amount of APV I use or also include Mg++. Thanks.

Two thoughts occur to me - the first is: How long have you had your 293s up?

The second is: when did you last do a prep of your plasmid DNA?