electroporation of LCLs

[Thierry Nouspikel]

In article <2i0uu4$kd1 at mserv1.dl.ac.uk>, brett at borcim.wustl.edu writes:
> Anyone transfected lymphoblastoid cells (LCLs) using electroporation?
> brett at borcim.wustl.edu

I did (and am still doing it). I'm using the following protocol:

- Resuspend cells (grown at 800000 to 1Mo/ml) at 10 Mo/ml in RPMI (no FCS)
  supplemented with 400 ug/ml E.coli tRNA as carrier, and 15-20ug/ml plasmid.
  Dispense 0.8ml/cuvette (at 37 oC).
- Use a Biorad GenePulser set at 250-260V, with capacity extender at 960 uF
  This gives time constants around 15 ms.
- Quickly transfer the content of the cuvette to 6 ml prewarmed culture medium
  (I use RPMI + 10-15% FCS, for transfection I add ITSS and ~20% old sup).
- Expect >50% dead cells after 24h. Cells can be passed at 300000 within 48h.
- Select... (but this is another story).

Hope this helps,

-- 
"Un technocrate, c'est un mec,	  !	Thierry Nouspikel, MD
 tu lui poses une question,	  !	Genetics and Microbiology Department
 quand il a fini de répondre	  !	CMU, University of Geneva
 tu comprends plus ta question!"  !	1211 Geneva 4, Switzerland
				  !	E-Mail:NOUSPIKE at cmu.unige.ch
 Michel Colucci dit "Coluche"	  !	Fax: 41/22/329.34.04