Transfecting Primary Cell Lines

Emmanuel Skoufos, Ph.D. skoufos at nefeli.imbb.forth.gr
Thu Mar 2 12:48:49 EST 1995


dcox at medmicro.uct.ac.za (Dermot Cox) writes:
>We are at present having problems transfecting primary cells derived from 
>foetal bovine testes with plasmid DNA.  We are using the DOTAP kit from 
>Boehringer-Mannheim with little success.  Has anyone had similar problems with 
>this kit or could make suggestions of alternate methods.

I am not aware of the kit you are using.  I successfully transfected primary
chicken cells using calcium phosphate precipitation.  Electroporation has 
been favored by some people also (I used it in cell lines and not with primary
cells).  In my experience, primary cells are awfully bad to transfect with
very low efficiencies, so don't expect stellar results.  Another thing you may
wnat to consider is the quality of DNA you use.  In my hands, transfections
work better when the plasmid is highly supercoiled (> 75%) and free of RNA.
Good luck with your venture.
Emmanuel
_______________________________________________________________________________

Emmanuel Skoufos, Ph.D.
Insect Molecular Biology Group
Institute of Molecular Biology and Biotechnology
Box 1527 
Heraklion, Crete 71110
Greece



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