Hi all,

I am currently trying out the Neon Transfection System to stably transfect my JEG-3 choriocarcinoma cells. So far I am getting issues where pretty much all the cells die after transfection i.e. none are attaching to the plate. My current protocol is:

1. Remove culture media, wash cells with DPBS, trypsinise.

2. Neutralise trypsin with media, count cells, then spin down at 200xg for 5 mins.

3. Remove media and wash cell pellet with DPBS. Spin again.

4. Remove DPBS and resuspend in Buffer R at 10 million cells/mL.

5. Mix 20ug DNA plasmid with 100uL cells in Buffer R. Take up into 100uL Neon tip.

6. Electroporate at 1600V, 20ms, 2 pulses. Immediately replate into warm culture media w/o antibiotics.

7. 48h later, change media and add media with antibiotics.

**Usually by this stage all cells are dead anyway**

Can anyone please help me work out what's going wrong?