Method: Electroporate COS-7

klenchin at facstaff.wisc.edu klenchin at facstaff.wisc.edu
Thu Nov 21 17:06:45 EST 1996


In article <Pine.PMDF.3.91.961121115708.25159D-100000 at OPAL.TUFTS.EDU>,
   jstrassw at OPAL.TUFTS.EDU wrote:
->Hello!
->After a LONG time trying to get high ewfficiency transfection of COS-7 
->cells, I finally got 50% transiently transfected with electroporation.  
->CaPo4 only gave a few percent as did many lipofection methods.
->
->So, here it is:
->3-5 million cells dont bother splitting berfore transfection
->0.4 cm gap cuvettes, 1050 microfaradays 220 mV
->10 micrograms of plasmid (Quiagen tip)
->Do it at room temperature and in complete medium, no pre incubation, no 
 
->post incubation. Dump the mess onto 100mm dishes with 10 ml of complete 
->meduim.  Wait 6 hours or over night.  Change medium.  THen adday as you 
wish.

You forgot to mention a crucial parameter: volume. Without it, you 
conditions are essentially useless. Also, I dare to suggest that 
postincunation for ~ 20 min at rt after dilution into complete medium is 
better than dumping cells in prewarmed medium right away. 


->Now, will someone post a decent transfection method for primary human 
->fibroblasts?

Same as above but you'll have to carefully optimize all the conditions. 
(Unless there is a very-very magic lypofection protocol).


- Dima



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