electroporation of PC12 cells - (Jan/06/2006 )
has anybody a working protocol for electroporation of NGF-pretreated PC12 cells? I am using the BTX 6000 device and the protocol works only one out of 15 experiments. A lot of cells are dead after transfection and transfection efficiency is very low.
How much DNA is still OK, meaning not getting too toxic for the cells? I tried from 5 to 50 µg and observed no changes...
Any information is very welcome since this is really getting on my nerves
I have no specific protocol for these cells, but you have to know that for most electroporations you will observe a lot of dead cells.
Browsing through literature on MT-4 cells, I have found about as much different electroporation conditions as there were different labs performing them... (some perform in PBS, others in RPMI, some in RPMI + DTT, some did at 300V, 960 V, 1050V, 1250V, 1500V, the capacitance values differed, the number of cells used differed, putting cells on ice or not and so on...).
On the other hand, most people used 10-12 µg of DNA for 2,5 million to 5 million cells...
Anothter thing about toxicity: make sure your DNA is very pure (endotoxin free plasmid preparations help a lot for transfections) and also that it is dissoled in MilliQ-H2O (if you get your DNA with precipitation or so: try to get rid of as much salt as possible).
Hope this helps a bit.