Electroporation cuvette gap size - Why?

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Fraser Moss
Fraser Moss's picture
Electroporation cuvette gap size - Why?

Commercial cuvettes come with different gap sizes between the electrodes.

Typically a cuvette for bacteria will have a gap of 1mm

A cuvette for mammalian transefction will have a gap of 2mm or 4mm.

I appreciate that field strength plays an important role in successful electroporation and the gap distance has a bearing in field strength

Ec=Vc/(0.75 x dcell)

Where Ec= Critical field strength (V/cm)
Vc = Permeation voltage of the membrane (1V @ 22 deg C and 2V @ 4 deg C)
dcell= diameter of cell

AND

For calculating the voltage to be set on the electroporator, the field strength Ec is multiplied by the gap width of the
cuvette.
V = Ec x dcuv

V = Ec x dcuv
V: Voltage (setting on Multiporator) [V]
Ec: Critical field strength [V/cm]
dcuv: Electrode distance (cuvette type) [cm]

This means at room temp Ec of a 20uM cell will be 667 V/cm

and in a 1mm cuvette voltage required for poration will be 66.7V
and in a 2mm cuvette 133 V and 4mm cuvette 267 V.

I therefore ask why do people use 2mm and 4mm cuvettes for mammalian cell electroporation and not use 1mm cuvettes and lower voltages?

Does it have something to do with increases in resistance (which would reduce current for a give voltage [I=V/R]) with the smaller gap between the electrodes? Or is it just because to electroporate 500000 mammalin cells takes a larger volume than 500000 bacterial cells?

Elizabeth
Elizabeth's picture
frasermoss wrote:

frasermoss wrote:

I appreciate that field strength plays an important role in successful electroporation and the gap distance has a bearing in field strength

Ec=Vc/(0.75 x dcell)

Where Ec= Critical field strength (V/cm)
Vc = Permeation voltage of the membrane (1V @ 22 deg C and 2V @ 4 deg C)
dcell= diameter of cell

Your post was very useful to me, so thank you, but I have a question about the formula used for calculating Ec. The Eppendorf website http://www.eppendorfna.com/faqs/CellBiology.asp gives a similar formula, but it has a factor 10 difference. They say: Ec = Vc/(15*a) in which a=radius of cell in cm. After adjusting the radius to diameter, you're still left with a factor 10 difference between the two formula's. So I thought you might have meant diameter of the cell in mm but in your example you give the diameter in cm....which formula is correct?

Jason King
Jason King's picture
frasermoss wrote: Or is it

frasermoss wrote:

Or is it just because to electroporate 500000 mammalin cells takes a larger volume than 500000 bacterial cells?

I think so. It can also be quite difficult to remove eukaryotic cells from a 0.1cm cell - you need to use yellow tips and following electroporation clumps can form which then sheer in the tip. I prefer to use 0.4 cm and a blue tip.