Hi all, for the past 6 weeks i have tried and failed to transfect JurKat cells, first i tried with pNL1.2 vector as it is required for the assay im working on, later i switched to pIRES-eGFP vector. I tried using Lipofectamine 3000 but it did not work and caused alot of cell death. Later i tried electroporation first with Amaxa II and now with BioRad GenePulser II. Each time no GFP positive cells are present, assesed through fluorescent microscope and westernblot. The pIRES vector has succesfully been used before by colleugues, altough in different cell types. Perhaps the pIRES vector does not work with JurKats? Or maybe there are some things im missing? The BioRad GenPulser II was succesfully used to transfect JurKat cells before in the lab (1989 so it was a while ago) but using the same protocol (280V, 950uF, 5*10^6 cells, 20ug DNA, 400 - 800 ul total volume) no transfection takes place.
Using viral transfection or purchasing new transfection reagens specifically for this is not really an option for the lab so guess im kinda stuck.
Does anyone have any tips for transfection of JurKats? Maybe some things im missing or new techniques/controls i can use?
All help is appreciated, and thanks in advance!
Check the paper by Greenlland et al Oncogene
. 2001 Nov 1;20(50):7386-97.
doi: 10.1038/sj.onc.1204870.
Hello everyone,
After a while of experimenting, I settled on a protocol that nets ~60-50% viability and ~60% of GFP-positive cells using electroporation. Figured I'd share it so that anyone having trouble transfecting Jurkats can give it a try. I am using the Biorad GenePulser II, using an exponential decay setting, at 400V and 300uF. I use 5*10^6 cells, resuspend them in 750ul ice-cold RPMI medium, and add them in a 4mm cuvette + 20ug DNA. After the transfection the cells are very fragile, so I use a glass pipette with a long neck and gently transfer them to 4ml pre-warmed RPMI medium in a 6-well plate.
Hope this helps!
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