The Multiporator®, in combination with the specially designed electroporation buffers, is optimally balanced for efficient and gentle electroporation of eukaryotic cells. Soft Pulse technology applies extremely short electric pulses for the highest survival rates. The relevant parameters of voltage and pulse duration are directly set, and the patented electronic pulse discharge ensures that they will be maintained exactly—independent of the sample resistance—for reliable and reproducible results.

What is the procedure for establishing a new protocol for the electroporation of eukaryotic cells?1. Check whether the cells will tolerate the hypoosmolar buffer. After 30 minutes' incubation, 90 % of the cells should survive, otherwise mixtures of hypoosmolar and isoosmolar buffer will have to be prepared until the desired survival rate is achieved. 2. Cell size can be used as a guide to the voltage to be used. A good approach is to make a rising series of voltages in 50 V increments. The time constant is usually 40 - 100 µs. Detailled information are included in the "General Protocol" and "Optimization Protocol" or in the Basic Application Manual for Electroporation which can be found on the Eppendorf Homepage (www.eppendorf.com / Support / Technical Documents / Manuals or Applications / Cell Technology - Instruments).

Can the Multiporators be upgraded with the module for bacteria or the electrofusion module?Yes, all Multiporators can be upgraded with the respective upgrade kits. Please note that the module has to be installed by a service technician.

Can external electrodes be connected to the Multiporator?Yes, this is possible via the "insert for connecting external electrodes" (order no. 4308 021.004). Modification of the Multiporator is not required for this. The external electrodes can be used both for electroporation experiments (all Multiporator models) and for electrofusion (devices with a fusion module). The insert has a function switch to adapt it to the method selected. The external electrodes are connected to the insert via 4 mm all-insulated lab plugs.

Can the resistance and/or the capacity of the Multiporator be set?No, these values cannot be set in any of the Multiporator modules. The advantage of the Multiporator is the fact that the time constant which results from capacity and resistance can be input directly and this results in the microprocessor-controlled pulse.

Why is it advantageous to use the Eppendorf hypoosmolar electroporation buffer for the transfection of animal cells with the Multiporator?In combination with the device, the buffers form a perfect system for electroporation experiments. They not only have low conductivity and an ion composition adapted to the inner cell environment; the hypoosmolar buffer in combination with Soft Pulse also facilitates gentle transfection. Under hypoosmolar conditions the cells swell as a result of water uptake. Because of the increasingly large size of the cell and the loosened cytoskeleton, electroporation can take place at a lower voltage. Furthermore, the cells assume a uniform spherical shape, which enables an effective optimization of the pulse parameters. A test of the cells for compatibility with the buffer must be performed beforehand. In addition, it is necessary to ensure that the cells are not subjected to hypoosmolar conditions for more than 30 minutes.

To what maximum pulse count can the Multiporator be programmed and what is the time interval between the pulses?The maximum number is 99, with an interval of 60 seconds in each case.

The electrodes of standard electroporation cuvettes are made of aluminium. Is there a danger that cytotoxic aluminium ions are released during the pulse and cause damage to the cells ?Pulses in the millisecond range intensify the hydrolysis of water, causing a steep pH gradient to form between the electrodes. Under acidic conditions, particularly aluminium is dissolved. The resulting high concentration of aluminium can have a very detrimental effect on the survival rate. With the microsecond pulses of the Multiporator, this effect no longer occurs. Literature: Friedrich et al, Bioelectrochemistry and Bioenergetics 47 (1998), 103-111

Is it possible to change the pulse duration of the Multiporator or is it fixed?The pulse duration (time constant) of the eukaryotic module can be freely selected between 15 and 500 µs in 5 µs increments. The pulse duration of the bacteria module is permanently set to 5 ms.

Is it necessary following the electroporation to incubate eukaryotic cells on ice for 10 minutes, as is frequently recommended in the protocols?As a rule it is not necessary to cool down the cells with ice following the electroporation. At 4 °C the "resealing" of the membranes is delayed, so that over a longer period of time an exchange of molecules takes place between the cell and its environment. This causes increased stress and can reduce the survival rate. When the closing of the membranes is to be slowed down intentionally e.g. to introduce large molecules), the cells should be cooled down with ice for a maximum of two minutes and then immediately transferred to a temperature of 37 °C.

Can PBS or medium be used as the electroporation buffer with the Multiporator?No, this is not possible. Since the conductivity of the PBS or cell culture medium is relatively high, this would result in too high a current flow. This would cause massive damage to the cells. The Multiporator is optimized for buffers with a low conductivity, therefore Eppendorf buffers should be used for the electroporation of eukaryotic animal cell.

Can the protocols for the Electroporator 2510 (bacteria and yeasts) also be used for the Multiporator?These protocols cannot be performed on the basic Multiporator model (eukaryotic module). However, they can be used with the optional bacteria module of the Multiporator.

Are the microsecond pulses of the Multiporator sufficient to rupture the membrane ?Yes. Rupture of the membrane already takes place after about 15 µs. The remaining time is needed to widen the "pores". Pulses in the millisecond range can cause massive damage to cells, e.g. due to electrophoresis.

What is the composition of the Eppendorf buffers used for electroporation and electrofusion?The buffer components are listed in the relevant Basic Applicaton Manuals for electroporation/electrofusion.

What is the maximum number of eukaryotic cells which can be used for an electroporation ?We recommend using a cell concentration of 1 x 106 cells/ml. In this range, the electric field can still efficiently influence the cells. At higher cell densities (3 x 106) homogeneous field conditions are no longer ensured, i.e. the cells are no longer subjected to a uniform electric field. This can lead e.g. to a decrease in the transfection rate. Using a cell density 1 x 106 cells/ml should have no effect on the transfection rate.

What material is the cuvette stand made of and can it be autoclaved?The cuvette stand for electroporation cuvettes is made of polypropylene and can be autoclaved at 121 °C.

What devices does Eppendorf supply for electroporation?1. The Electroporator 2510 and the new Eppendorf Eporator are especially suitable for porating bacteria and yeasts. The time constant permanently set at 5 ms allows new protocols to be optimized simply by selecting a pulse voltage between 200 and 2,500 V. 2. In combination with the electroporation buffers, the Multiporator makes an ideal system for transfecting eukaryotic cells. In combination with the hypoosmolar buffer, the µs pulse (Soft Pulse) which handles cells gently efficiently transfects cells at a simultaneously high survival rate. The microprocessor-controlled pulse discharge facilitates optimization by directly programming the time constant and thus guaranteeing the reproducibility of experiments. The Multiporator is available or can be upgraded with two optional modules. The bacteria module can be used to perform electroporation on bacteria and yeasts. Equipping the module for cell fusion involves a Micro fusion chamber to optimize parameters and a Helix fusion chamber to perform the real fusion experiment.

In which solution purified DNA should be soluted in order to use it for electroporation?DNA should be soluted in very pure water. Compared with TE buffer, the results using aqueous solutions of DNA are substantially better. Especially EDTA, even in µmol concentrations, is strongly toxic as a complexing agent in the cell. Both electroporation buffers from Eppendorf can also be used to dissolve DNA, however these buffers should not be used for the elution of DNA in nucleic acid purifying kits.

How can bacteria be transformed successfully using ligation preparations?Ligation preparations generally include reaction buffers containing salts to increase the conductivity of the sample. Since this may affect the electrical parameters of the device and thereby can lead to a lower transformation rate, the conductivity of the ligation preparation should be reduced by one of the standard methods below. 1. Precipitate the ligated DNA by adding ethanol or butanol and glycogen as described in Biotechniques 16, 988. 2. Dilute the ligation preparation with water.