Hello!

I`m currently trying to transfect the jurkat cells with plasmid DNA. I`ve already tried lipofectamin and electroporation but niether of the two worked.(( I really need some fresh idea!

[quote=Elena Lidlein]Hello! I`m currently trying to transfect the jurkat cells with plasmid DNA. I`ve already tried lipofectamin and electroporation but niether of the two worked.(( I really need some fresh idea![/quote] I haven't done this for over 10 years so I'm sure there will be some better transfection reagents available now (eg. Lipofectamine 2000 from Invitrogen, ExGen500 from Fermentas etc). I would check whether these reagents have been used on Jurkats. If not then here's my old protocol which gave about 5% efficiency which was OK for me). Wash the cells twice with 1 x TBS Prepare and combine the following solutions: 250L TBS containing DNA 250L TBS containing DEAEdextran and chloroquine (25L of a 10mg/mL DEAE-dextran stock and 2.5L of a 4mg/mL chloroquine stock) Resuspend 10-15 million cells in the DNA/DEAEdextran solution Incubate at RT for 30 mins, with occasional mixing Add 3 mL of TBS and spin down the cells (300g for 4 mins) Wash the cells with complete medium once Add fresh medium (32mL: 450000 cells/mL) Incubate at 37 degrees for 24 hours PHA/PMA induction Harvest cells 48 hours later TBS= 25mM Tris 7.4, 137mM NaCl, 5mM KCl, 0.7mM CaCl2 (H2O)6, 0.5mM MgCl2 (H2O)6, 0.6mM Na2HPO4 ....and I can't for the life of me remember why or whether I did the PHA/PMA induction. Maybe you don't need to. Also, I have no note of how much plasmid DNA was used. Sorry. It must be quite a lot for this number of cells though. Maybe someone else has some info on this - otherwise you might need to scale the transfection process down and titrate with varying amounts of plasmid DNA. Good Luck!

Thank you so much for the protocol you`ve sent!
The thing is that I`m using the dual luciferase assay to cheque the enchancer activity. So I`m afraid that the PMA induction is no good in my case.