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Electroporation F.A.Q

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Electroporation

What is electroporation?

Electroporation is the process in which a cell is subjected to an electrical current surge (pulse). This pulse creates temporary openings (pores) in the cell membrane, which allow molecules or particles to enter the cell. These molecules come in various different forms, including dyes, proteins, DNA, RNA, and plasmids.

Why is electroporation a useful transfection technique?

Electroporation enables the transfection of large numbers of cells (1.106 cells/ml) in an electroporation sample, which leads to a high transfection efficiency. In the case of mouse fibroblasts, a transfection efficiency of over 40 % of all cells is possible. Several cell lines that are difficult to transfect (e.g. ES cells) can only be processed by means of electroporation. Electroporation can be used for eukaryotic cells, plant, and animal cells as well as for bacteria and yeast.

What types of electroporation are carried out?

The electroporation of bacteria is carried out using a high voltage and relatively long pulses (approx. 5 ms). When electroporating eukaryotic cells, it is necessary to differentiate between electroporation with long pulses and high conductivity and electroporation with short pulses and low conductivity. This is possible using the Multiporator electroporation system.

What is the maximum sample volume I could use with the electroporation cuvettes?

The maximum sample volume depends on the cuvette size. The maximum samples sizes for the various size cuvettes are as follows:
1 mm gap: 100 µl
2 mm gap: 400 µl
4 mm gap: 800 µl

What advantages does the Multiporator offer?

Conventional devices use long pulses with a relatively low voltage and a highly conductive electroporation medium (usually PBS, sucrose solution, or a growth medium). In the case of the Multiporator, very short pulses are combined with a relatively high voltage. Using Eppendorf’s hypoosmolar buffer, which has low conductivity, can reduce the voltage to a value that is well tolerated by the cells. This enables electroporation of the cells to be performed in a much more “cell-friendly” manner. Therefore an overall increase in efficiency and survival rates is achieved.

What are the time intervals between the individual pulses in the Eukaryotics mode of the Multiporator?

One minute elapses between each pulse.

How is electroporation regulated?

The Multiporator regulates the voltage impulses for the electroporation of eukaryotic cells in exact accordance with the set parameters. The nominal and actual values for the respective voltage applied are controlled in short intervals and are regulated permanently. Only the relevant parameters voltage (V) and time constants (ps) – need to be entered. This means that specific capacitors and resistances no longer need to be aligned to specific voltages.

Which buffers are used for electroporation?

The use of electroporation buffers from Eppendorf with low conductivity and low osmolarity is advantageous for the following reason. Low osmolarity enlarges the cells and thus facilitates the breakdown of the membrane and the formation of membrane pores. Low conductivity enables pulses with a lower field strength (voltage) to be used.

Due to their relatively high conductivity and osmolarity, commonly used electroporation buffers, such as growth media, PBS and sucrose solution, cause a relatively high current to flow through the cuvette over long periods of time, which may damage the cells. This does not occur when the Eppendorf electroporation buffers are used with the Multiporator.

How does the hypoosmolar buffer affect electroporation?

The hypoosmolar buffer creates osmotic overpressure in the cell during electroporation, which causes water to enter the cell. The cell enlarges and the membrane expands. This leads to a reduction in the voltage applied. Furthermore, the swelling of the cell causes the cytoskeleton to break, which leads to the membrane losing its internal stability. This facilitates the electroporation of the cell and increases the efficiency of the application.

Is it possible to use other buffers with the Multiporator?

Only the special buffer system from Eppendorf, consisting of one hypoosmolar buffer and one isoosmolar buffer, together with the Multiporator ensures best results. The buffers fulfill high purity criteria, which are crucial for experiments using sensitive eukaryotic cells. They are sterile when bottled and free of mycoplasma and pyrogens, which could reduce the survival rate of the cells when present. The buffers can be transported and stored at room temperature (max. 65°C) and have a shelf life of one year. The buffers and device combine to form a closed system. The use of other buffers is not recommended.

Why do large cells require lower voltage?

The field strength applied depends on the size (i.e. the radius) of the cell. This correlation can be approximated using the following formula:

E =Vc
——
15*a

Ec: Required critical field strength [V/cm]
Vc: Critical breakdown voltage [V]
a: Cell radius, measured in electroporation buffer [cm]

The voltage for the breakdown of the membrane is virtually identical for all cell lines (1 V at room temperature, 2 V at 4°C). If the cell diameter is doubled by the hypoosmolar buffer, the voltage applied to the cell is doubled and the voltage through the cuvette remains the same. This means that the breakdown voltage may be attained even if the applied voltage is halved.

How is efficiency increased?

The required field strength can be estimated with the aid of the functional correlation of field strength and cell radius. This should be regarded as the starting value for optimizing field strength for the cell line used in the respective buffer. This theoretical value is normally the lowest for the voltage series tested, which leads to a marked increase in efficiency.

How does the temperature affect electroporation?

The influence of the temperature has two contrasting effects. On the one hand, the temporary pores created by the pulse close much more slowly at a low temperature (4°C), because the membrane lipids have sunk below their melting point and the material has more time to penetrate into the cell. This is one reason for using the lower temperature of 4°C, particularly in the case of material with a low concentration. On the other hand, the vitality of certain cells (e.g. Jurkat) decreases rapidly under hypoosmolar conditions at a low temperature. In this case, electroporation should be carried out at room temperature.

To what extent does the pulse length affect electroporation?

The longer the pulse exerts an effect on a cell, the greater the risk that the cell will be damaged by the voltage applied. Localized heating of the cell leads to irreparable damage, and electrophoresis causes substances to flow into the cell and the contents of the cell to flow out of the cell (e.g. the collapse of the Na+/K+ gradients of the cell). To avoid this effect, extremely short pulses are used with the Multiporator (µs range instead of the ms range used with conventional electroporation systems).

How important and how necessary is it to vary the number of pulses?

For many cell lines, electroporation is performed as a single pulse technique, which guarantees high transfection rates. However, if this single pulse fails to produce satisfactory results, it is also possible to carry out multiple pulsing. There is a 60-second interval between the pulses, which ensures the reforming of membrane pores. Due to Brown’s molecular movement, the cells can rotate between the pulses. This enables additional pores to be created in other areas of the cell membrane.

Why does mycoplasma cause interference during electroporation?

Mycoplasma grows on the surface of the cell membrane and thus hinders the build up of voltage necessary for the formation of pores. The cell lines should therefore be checked for mycoplasma contamination.

Why should trypsin not be used during electroporation?

Trypsin is normally used to release adherent cells from the surface of the culture dish. However, the enzyme damages the cell membrane to such an extent that the cells are then too sensitive for the electroporation procedure. This causes low survival rates and low-quality reproducibility of the transfection result. For the “cell-friendly’ removal of adherent cells, it is therefore advisable to use dispase instead. Dispase can be used with some cells in the growth medium (e.g. in the case of L-cells) or if release is lower in PBS without Ca2+/Mg2+ (e.g. with BalbC 3T3). Should suspension of adherent cells not be possible with the aid of dispase treatment, the cells may be removed mechanically using a cell scraper, which is also more “cell-friendly” than trypsin treatment.

From which manufacturer can I order the dispase recommended in the instruction manual of the Electroporator instead of trypsin?

The dispase is available from Gibco (Art. no. 17105-04). This involves a lyophilisate that must be subjected to sterile filtration after dissolution. In sterile form, the dispase is available from Roche Diagnostics.

Why should a medium be used which does not contain phenol red?

Phenol red has a toxic effect upon entering the cell. For this reason, cells should only be treated with a phenol-red-free medium prior to and following electroporation. A medium with a phenol red content may be used for cultivation purposes 48 hours after the end of electroporation.

Which buffers are used for storing the DNA to be transferred?

Following isolation, the DNA should not be diluted in buffers with complexing agents, as even low concentrations of these agents can severely affect the cellular metabolism of the electroporated cells. Instead, dilute the DNA in isoosmolar buffer.

How are the cells removed from the cuvette?

Cells should not be removed immediately; they often require a “rest period” of 10 minutes in the cuvette. Electroporated cell suspension must be removed carefully. The cells are stressed as a result of electroporation, so care must be taken to ensure that the cells are not exposed to turbulence or high flow rates, as is the case during pipetting with narrow pipette tip openings and rapid piston movements. Removal using a pulled Pasteur pipette has proved to be very successful. Any small amount, which may remain in the cuvette, does not affect the result.

What would happen if arc formation occurs and the lid springs open inside the device?

The Multiporator has been constructed so as to prevent any arc formation strong enough to cause the cuvette lid to spring open. Current-limiting resistance and newly developed electronics prevent such high currents in the device when Eppendorf buffers are used.

Does condensation on the cuvette affect the time constant?

No. However, the cuvette should nonetheless be wiped before being placed into the cuvette holder.

What material is used for the cuvettes and electrodes?

The cuvettes are made of polycarbonate and the electrodes are made of aluminum.

How is it possible to identify the different gap widths?

All Eppendorf cuvettes have blue lids. The gap width and the maximum volume are printed on the side of the cuvette as well as on the individual packaging. Recently, I failed to transform my bacteria with DNA obtained from a ligation experiment.

How can bacteria be successfully transformed with ligation mixtures?

Ligation mixtures generally contain salts from the reaction buffer. This will affect the time constant and may lead to reduced transformed rates. Therefore we recommend reducing the ionic strength of the reaction mix after ligation with one of these two common methods:

  1. Precipitate the ligated DNA using ethanol or butanol and glycogen as described in Biotechniques 16, 988.
  2. Dilute the ligation mixture with water.
Can electroporation cuvettes and lids be cleaned and re-used?

Theoretically, electroporation cuvettes can be reused. They can be cleaned in water or alcohol, and then dried and autoclaved. However, after a few cycles, the polycarbonate will begin to show stress cracks and leak liquid. Due to the high currency and temperature in a sample, it is possible that traces of cells and DNA will stick to the surface of the electrodes. This may lead to contamination or reduced time constants. In addition, inhomogeneous contact of the suspension to the electrodes may arise. Therefore we do not recommend reusing the cuvettes. It can not be guaranteed that they will survive multiple autoclavings or that they will be sterile in subsequent uses. In addition, the lids are not autoclavable and will melt.

How do sample volume and buffer resistance affect time constants with the Electroporator 2510?

The time constant of 5 ms is determined by a 10 mF capacitor and a 500 ohm resistor (assuming a high resistance buffer, approx. 3,000 ohm). The sample volume does not significantly effect the time constant as long as the sample has a high resistance. Cell suspensions up to 800 ml can be transformed with the Electroporator using the Eppendorf cuvettes with gap size of 4 mm. The gap size is important for calculating the necessary field strength (V/cm) inside the sample. A sample of low resistance will reduce the total resistance of the system and therefore reduce the time constant significantly.

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Microinjection/Micromanipulation

How can I determine what volume I am using when I do cell injections with the Transjector?

The volume injected is not displayed on the Transjector. However based upon the programmed injection pressure and time, the injection volume can be calculated. The following reference discusses how to do this calculation.

Quantitation of the Volume of Liquid Injected into Cells by Means of Pressure
G. Minaschek, J. Bereiter-Hahn, and G. Bertholdt
Experimental Cell Research 183 (1989) 434-442

I want to use my own capillaries with Eppendorf micromanipulators and microinjectors. Which capillary grip would fit my tips?

All Eppendorf microinjectors (Transjector, FemtoJet, and CellTram) are equipped with the Universal Capillary Holder. With this holder the different grip heads can be interchanged for use with different size capillaries. Use the table below to determine which grip head suits your needs.

DescriptionOrder No.
Capillary grip 0: fits microcapillaries with an outer diameter of 1.0 to 1.1 mm920007414
Capillary grip 1: fits microcapillaries with an outer diameter of 1.2 to 1.3 mm920007708
Capillary grip 2: fits microcapillaries with an outer diameter of 1.4 to 1.5 mm920007716
Capillary grip 3: fits microcapillaries with an outer diameter of 0.7 to 0.9 mm920007724
Can the cardboard box in which the Microloader is dispatched be inserted into the autoclave when the Microloader is sterilized?

Yes, it can be autoclaved once.

What are CELLocates made of?

CELLocates are made of glass.

What are the dimensions of CELLocate?

The diameter is 12 mm by 12 mm, with a thickness of 0.17 mm �0.04 mm. CELLocates are available in these dimensions only.

Which headstage types (amplifiers) fit into the headstage holder (amplifier holder) on the PatchMan micromanipulator?

The following amplifiers fit into the PatchMan headstage holder: HeKa EPC 7/EPC 9 and Axon CV-4, CV-5, HS-2, and HS-2A

Can the angle of injection of InjectMan be varied to suit different applications?

Yes. The axial angle of movement can be adjusted for both semi-automatic injection and manual injection with the Axial function. The adjustable angles are stated in the operating manuals and vary according to the software version used (15–45° or 75°). For injection in angles not equal to 45°, the angle on the motor head must be adjusted accordingly in order to guarantee axial injection movement.

My Micromanipulator 5171 is currently mounted on an old Zeiss microscope with the aid of the universal clamp. However, I now wish to mount the micromanipulator onto an Olympus IMT-2. Is there anything that I should pay particular attention to?

A special adapter (5171 055.000) is available for mounting all Eppendorf micromanipulators onto the Olympus microscope IMT-2. It is essential that this adapter be used. The universal clamp (5171 045.004) is not necessary and should be unscrewed completely from the motors. As soon as the special adapter is attached to the Olympus microscope, you can start mounting Micromanipulator 5171 directly onto the adapter. Detailed instructions on how to mount the micromanipulator onto the microscope can be found in the operating manuals for the device and the adapter.

What can you recommend as regards sample preparation of the injection solution for microinjection?

Our recommendation:

Samples should always be centrifuged (for 15 minutes at maximum speed in a microcentrifuge) immediately before the capillaries are loaded.

Use the Eppendorf Microloader for filling Femtotips (backloading). Use only once. The liquid should be extracted from the top of the tube. Make sure that no gas bubbles are in the glass capillary after loading has been completed. If your solution contains proteins, you should work as quickly as possible after the capillary has been loaded. If the injection solution is not introduced into the medium immediately, there is a possibility that the injection solution will dry in the capillary tip, thus blocking flow.

What is the difference between dynamic and proportional movement?

Dynamic movement was originally developed for adherent microinjection and enables extremely stable positioning of the capillary. In the case of dynamic movement, the direction and speed of the tool movement are controlled by the joystick. TransferMan 5177 and InjectMan are dynamic systems.

In the case of proportional movement the position of the tool is controlled by the movement of the joystick (in a similar manner to a PC mouse). Our TransferMan NK and many hydraulic/mechanical systems operate proportionally.

We usually recommend proportional movement for work involving suspension cells, with dynamic movement being preferred for adherent microinjection. The dynamic mode can also be used for suspension cells, depending on the experience of the user and on the task at hand.

With which devices is it possible to generate a vacuum that can hold difficult-to-manage cells (e.g. plant cells)?

Suspension cells are best held using CellTram Air. If cells need to be firmly fixed in place on the holding capillary, CellTram Oil can be used instead of CellTram Air (e.g. for biopsy or difficult-to-hold cells).

Is it possible to reduce the length of the tube that is mounted onto the universal capillary holder or Transjector/FemtoJet from two meters to one meter?

Yes, however, attaching the tube to the capillary holder causes a problem. Since the tube is mounted to the universal capillary holder or Transjector / FemtoJet with the aid of a special screw attachment, this screw must also be fastened to the shorter tube. For safety reasons and to prevent leakage, we do not recommend shortening the tube, as it has no applicable benefits.

Can FemtoJet be controlled externally, e.g. by a TTL connection?

Yes. The device can be controlled externally (e.g. an injection may be triggered) via an RS-232 interface.

How do I convert from hPa to Psi?

1 hPa = 0.0145 Psi

1 Psi = 68.97 hPa

Do you know of any special glass slides or Petri dishes that minimize cell adherence?

No, contact the manufacturers directly (e.g. Nunc, Nalgene, Greiner).

Do you have a publication which explains how to determine the diameter of microinjection capillaries and indicates the influence of the diameter on the injection volume?

Yes, we do have one such application: Experimental Cell Research 210, 260-267 (1994): Microinjection Technique: Routine System for Characterization of Microcapillaries by Bubble Pressure Measurement, available from our Application Support ([email protected]).

Do you know of any publications on injections into insect embryos?

Yes: Peloquin et al., 1997. Electromechanical microinjection of Pink Bollworm Pectinophora gossypiella Embryos increases survival. BioTechniques 22 , 496-499.

The authors claim that this application is based on injection techniques into insect embryos (Pectinophora gossypiella). They compare Narishge manipulators with the Eppendorf Manipulator 5171 and conclude that the survival rates obtained using Manipulator 5171 are five times higher!

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