klenchin at macc.wisc.edu
Thu Jul 13 15:10:50 EST 1995
In article <3u31k6$218 at sifon.cc.mcgill.ca>,
Graham Dellaire <popa0206 at PO-Box.McGill.CA> wrote:
>HAs anyone out there obtained an electroporation frequency
>better than 10-5 transformants/no# cells transformed???
Of course. In brief, for L,3T3, CHO 10e(-3) is readily achievable; for
Jurkat, K562, primary fibroblasts 10e(-4) to 10e(-6) is more typical (I
must say though that Ive never had a goal to get the highest numbers).
Ive done my Ph.D. thesis on the mechanism of electrotransfection
(a.k.a. electroporation). As I see a lot of electroporation-related
questions on this group, Ill summarize my experience and
recommendations here based both on practical and theoretical
The mechanism is electrophoretically driven DNA insertion through
electropores (see Klenchin et al., Biophys. J., 1991 and Sukharev et
al., ibid, 1992). Hence, the condition:
1. Pulse should be low amplitude/long duration exponent (pores
created are larger and live longer; long nonporative and less damaging
tail works for electrophoresis). Practical sense: use the highest
capacitor available and find optimal voltage (see below).
The probability for the DNA to get to the pore increases with increasing
both cell and DNA concentration. =>
2. Increase both. It all depends on exact situation but
generally, Id say, cells 1x10e7-5x10e7/ml and DNA 20-100 ug/ml.
An important distinction: are you interested in total number of
transformants obtained (*efficiency*) or you need *frequency* of
transformation to be high? Electroporation and, in part, DNA input
depend on voltage ~ exponentially, whereas concomitant cell death
dependence isnt that sharp. => For the former, one might want to go for
very harsh pulsing conditions killing 90+% of cells and high cell/DNA
concentrations. For the latter, you need to find a compromise between
DNA uptake and cell death. Practically speaking:
3. fix electroporation medium, suspension volume, capasitance,
choose cell and DNA concentrations and titrate voltage. Conditions
where 25-50% of cells survive (by plating and counting, not by Trypan
Blue! T.B. underestimates death dramatically) should cover window of
optimal frequency. It is important do do it *with* DNA as DNA increases
cell death (passing DNA enlarges electropores making some of them
4. Room temperature works better in 90+% cases.
5. It depends, but generally supercoiled and circular plasmids
work better for transient, while cut works better for stable
transfections. Effects here could be small or large depending on cell
line and plasmid itself.
6. Dont bother with more than one pulse.
7. No prepulse incubation of DNA is necessary.
Electroporation medium is a tricky issue. Effect on the level of
electricity is simple: in low salt the pulse is much longer then in high
salt (but in sucrose the same amplitude/duration gives lower frequency).
All other effects are on the level of cell survival. One can spend life
optimizing components of the buffer. Here is my approach to the problem.
I found the buffer that appeared to work equally well for a variety of
cell types (including even Drosophila cell line) and, in my hands,
better than everything else described in the literature.
Note: Ca2+ (100 uM and up) in electroporation medium is toxic
for fibroblast-like cells but has no effect on lymphocyte-like cells.
7. Standard PBS (sorry, dont remember protocol) is modified in
the following way:
- HEPES is used instead of phosphate (it is frequently referred to as
- K+ and Na+ are swapped (e.i., the solution is intracellular - high
K+, low Na+);
- add 2 mM Mg2+
- add 0.5% Ficoll 400 is added;
(Do not autoclave! Sterylize by filtration).
Ficoll at this concentration dramatically helps to survive (why? -
mystery) thus enabling to increase voltage and/or duration. We used to
call this buffer TrakhoDim (TD, after Dima).
8. While no postpulse incubation is necessary, 5-10 min after
pulse do not affect survival significantly. Cells, however, should not
be diluted right away into warm culture medium. Use rt medium and let
pores reseal for ~ 20 min before placing cells in CO2 incubator.
9. Bio-Rad pulser is very good. I dont however, see how any
other system that has the same voltage and capacitors could be worse.
If I were to set good electroporation protocol for my needs, Id do the
Stick to the pulser I already have. Use noncut RNA-free plasmid at 50
ug/ml and cells at 3x10e7 in 200 ul of the above electroporation medium.
At highest capacitor available, optimize voltage to find 35% survival
conditions, then repeat the same for transformation frequency/efficiency
around the foltage found with smaller voltage increments. Than stick to
the best voltage for the given cell line. Repeat the same for every cell
If you guys have any questions or if this will help in any way, please
email me - Im curious to know results.
Best of luck!
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