problem transfecting pc-3 cells - (Jan/23/2006 )

Hello there,

I am new to this forum but have found some very interesting info on it before now so thought I might be able to get some help.

I have been transfecting pc-3 cells with PCDNA3.1 + VECTOR and have got an enormous amount of cell kill infact most of my cells keep snuffing it!!!

I am transfecting with 2ug of pcdna3.1 +, 4ul plus reagent and 6ul of lipofectamine in serum free medium in a 35mm dish.


I dont have antibiotic in the media, the cells are a pretty low pass number, I have done a lipo only control and they grow absolutely fine.

I dont know what else to try I have even tried new and old stocks of vector as I did have them transfecting fine last year and of coure I need to do a similar expt and now it wont work!!!!

The cells have also been mycoplasma tested, transfecting with pegfpn1 does not seem to have any detrimental effect it only seems to be wih pcdna3.1.

Anybody got any tips??????????


Thank you for your time,

Kerry

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Decreasing the DNA amount may help. Recently I did pGL-3 vector transfection in PC3 cells using lipofectamin+plus reagent. I used two dosages of DNA, 4ug and 6ug for a 100 mm dish. 6ug caused much more cell death than 4 ug.

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Hi,

Thanks for your reply,

I have tried both 2ug and 4ug in a 35mm, and the 2ug caused less kill than the 4ug, but still a huge a mount of cell kill, but I might try less agin, I really think i is something to do with this particular vector as I have no problem at all with pegfpn1 and a couple of other vectors, but had really wanted to use PCDNA3.1 + for comparison studies from experiments in other cell lines, and to save a lot of cloning!!!!!

Thank you,

Kerry

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