Transfection of EBV-transformed B cell lines

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Sun Feb 9 22:59:48 EST 1997


In article <32FE6AE7.7123 at rex.re.uokhsc.edu>, kprillim at rex.re.uokhsc.edu 
commented on a "magic" problem of transfecting EBV-transformed cells:

#I use essentially the same protocol described by John, but with a few
#changes I've made (and either with these changes or with dumb luck, I
#begin to see distinct foci of transfected cells within 2 weeks). =

Well, call it my idiot ego, but I can't resist commenting (hope it'll help 
someone).  

#To begin, I will also give the reference for cytomix, which I agree
#works well.  The reference is: Nucleic Acids Research, vol. 20, no. 11
#(van den Hoff et al.).  The "recipe" they give is: 120mM KCl; 0.15mM
#CaCl2; 10mM K2HPO4/KH2PO4, pH 7.6; 25mM Hepes, pH 7.6; 2mM EGTA, pH 7.6;
#5mM MgCl2; pH adjusted with KOH.  

Jesus, someone even bothered to publish such a simplistic idea - 
electroporation medium keeps more cells alive if it resembles an intracellular 
conditions. Well, I bet the results will be better if one 1) avoids Ca/EGTA 
altogether, 2) decreases MgCl2 to 2 mM, 3) adds 0.4% Ficoll 400

#They suggest that, just prior to use,
#the cytomix be supplemented with ATP (2mM, pH 7.6 with KOH) and
#glutathione (5mM), but I do not add these supplements as I have found it
#unnecessary. 

Exactly. No need at all.

#3) Add 750 uL of the cell suspension to each electroporation cuvette (be
#sure to include cuvettes for "vector-only" and "cells-only" controls
#along with your experimental ones!).  To this, add 30 ug of the DNA (mine
#is usually in TE buffer) to be transfected (being certain that the
#plasmid volume does not exceed 50 uL, as the recommended maximum volume
#of the cuvettes is 800 uL); to the "cells-only" cuvette, add a similar
#volume of TE alone.  Mix the DNA with the cells by gently inverting the
#capped cuvette a few times.

Just by decreasing the volume 2-fold to ~ 400 ul, one achieves 1) higher cell 
and DNA concentration, 2) longer pulse duration. Both factors should improve
transfection effeciency.

#4) Incubate the cuvettes on ice for 10min.  

Use room temperature throughout!

#The electroporations are
#then carried out at 250V and 960 uF capacitance.  
                     ^^^^
Optimize for yourself for every cell line separately.

#Just prior to
#"zapping", mix the cells once more by gentle inversion and then
#electroporate.  You may want to watch and/or record the decay time
#during this: it should fall between 10-25msec (lower is better). 

*Higher* is better (provided that voltage is chosen properly).

#5) Following each "electrocution", mix the respective cuvette and place
#it on ice once again for 10min. 

Don't. Better dilute them without preincubation into rt medium and leave 
at rt for ~ 20 min. Only then place in the incubator.

#6) Begin selection on the third day.  Count each flask, pellet them, and
#then resuspend in selective media (for the "cells-only" flask however,
#split it equally and resuspend one half in selective media and the other
#half in non-selective media...this provides good visual clues for
#determining when "non-transfected cells should be dead").  The selective
#medium I use is 1.5mg/mL G418 (I go a little "heavy" I know, but I
                 ^^^^^^^^^^^^^
This *definitely* depends on the particular cell line, and must be 
predetermined. 

- Dima

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