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.

For this purpose, the cells are incubated in different mix ratios of hypoosmolar and isoosmolar buffer for 30 min. Starting from the hypoosmolar buffer, osmolarity should be increased in increments of approx. 50 mOsmol. The survival rate of the cells is then examined (e.g. using Trypan blue staining). Taking account of the fact that at least 90 % of the cells in the buffer must survive, the buffer with the lowest possible osmolarity should be selected. A mixing table is included in the Basic Application Manual for electroporation, for example.

The beaker and the core of the Helix fusion chamber should be rinsed thoroughly with distilled water immediately after the experiment to prevent drying of sample residues. If it is severely contaminated, the Helix fusion chamber can be cleaned briefly in an ultrasound bath (possibly with a cleaning additive like Edisonite-Super, for example) or with a very soft (tooth) brush. If using a brush for cleaning, you must ensure that you brush in the direction of the winding, otherwise the electrodes may be moved making the Helix fusion chamber unusable. Sterilization can be effected by treating with ethanol (70 %, non-denatured). To this end, the beaker is filled with 250 µl of ethanol and the core screwed into the beaker. After 10 seconds, the core is unscrewed and the alcohol can be removed. For subsequent drying, place the beakers and the core in the stand under sterile conditions (e.g. clean bench).

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 here.

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.

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. .

"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"

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.

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.

The buffer components are listed in the relevant application manual for electroporation/electrofusion.

The cuvette stand for electroporation cuvettes is made of polypropylene and can be autoclaved at 121 °C.

Yes. The specified cuvette volume (e.g. 100 µl for a 1 mm cuvette) is the maximum volume and also the optimum volume for the electroporation. Smaller samples can also be used, as long as the electrodes are connected through liquid. It is only necessary to be sure that the field strength during a pulse in the cuvette is less than 10,000 V/cm. Above this value, electric sparkover can occur through the air.

Yes, our Userguide No 0112/06 “Microinjection of plasmid DNA or double stranded RNA into the gonads of C. elegans” contains lots of useful information about this kind of application. The Userguide is available at our website.

Microinjections into embryos are normally performed manually with an injection pressure of 300-500 hPa and a very short injection time of 0.1-0.2 seconds. The high injection pressure prevents the injection needle from clogging.

- High speed for efficient penetration of rigid structures (Vmax 7,500 µm/sec) - Motors with high resolution for smooth step-free motions. - Resolution per step: 0.04 µm - Easy preset of speed or work area via control unit - Menu controlled programming - Storage of user profiles

Yes. The axial angle can be adjusted for semi-automatic injection and for manual injection with the axial function (e.g. into C. elegans). The adjustable angles are found in the operating manual and vary between 35° and 55° (positioned in the middle hole of the guide). For injection angles different from 45°, the angle settings must also be changed in the menu (Install/Angle).

Only one adapter is necessary.

In principle it is also possible to use the TransferMan NK 2 for injection into adherent cells. However, our InjectMan NI 2 is much more suitable for this application. The unique electronic coupling of the InjectMan NI 2 with the microinjector FemtoJet makes microinjection into adherent cells very quick and easy. The TransferMan NK 2 with its proportional joystick is specialized for the manipulation of suspension cells.

For the injection into plant cells, a pressure greater than 7,000 hPa is very often necessary. Therefore, a CellTram Oil or vario should be used for plants with a high turgor (max. pressure 20.000 hPa).

"According to the injectors in use you have to order the following tubes:
Connection to CellTram Air/Oil/vario: 5176 114.004
Connection to Transjector/FemtoJet/FemtoJet express: 5246 164.004"

Yes, this is possible, as both liquids usually do not come into direct contact.

CellTram vario has an additional fine drive. The transmission ratio between fine drive and coarse drive is 10:1. The minimum movable oil volumes are 0.02 µl (with coarse mode) and 0.002 µl (fine mode). The maximum pressure is 20.000 hPa for both devices.

Yes, that is possible. Please use an appropriate capillary.

For this purpose we offer a Service kit (5176 195.004) that contains an appropriate tool. Alternatively you can also use a thin metal wire.

"All Eppendorf microinjectors (Transjector, FemtoJet/FemtoJet express and CellTram) are equipped with the Universal Capillary Holder. Different grip heads can be interchanged for the use of capillaries with different outer diameters. Use the table below to determine which grip head suits your needs.
Description Capillary grip 0: fits microcapillaries with an outer diameter of 1.0 to 1.1 mm, order no. 5176 210.003
Capillary grip 1: fits microcapillaries with an outer diameter of 1.2 to 1.3 mm, order no. 5176 212.006
Capillary grip 2: fits microcapillaries with an outer diameter of 1.4 to 1.5 mm, order no. 5176 214.009
Capillary grip 3: fits microcapillaries with an outer diameter of 0.7 to 0.9 mm, order no. 5176 207.002 "

The typical injection volume ranges between 60-100pl

The injection volume depends on the set pressure and the type of capillary, as well as the residence time of the capillary in the cell. Furthermore, the injected volume depends on the type of cell and the solution to be injected. Due to the above described factors, it is not possible to give exact settings for a specific injection volume. The user always has the option of determining the injection volume for the present experiment by making a type of "calibration curve".
Possible methods of determining the volume:
1. Injection of an enzyme that is normally not present in the cell (e.g. luciferase) in 50 - 100 cells and determination of the injection volume by subsequent enzyme assay:
- Add pure luciferase (Sigma, final conc. 2 mg/ml) to the injection solution.
- Inject an exact number of cells with the FemtoJet/FemtoJet express with a known setting.
- Lyse cells and determine the luciferase activity from the extract with a luminometer (as described in the literature).
- Prepare a dilution series from the injection solution (2 mg/ml) and determine the luciferase activity in this series. - Plot a curve of the measured activity versus volume. - Read off the injected volume from this curve and the measured activity of the cells.
- Calculate the volume per cell from this read volume.
2. Injection of a defined radioactivity into one water drop (20 - 100 times), followed by measurement of the radioactivity (Geiger counter). The approximate injection volume can be calculated from the radioactivity of the solution.
3. Injection of fluorescence (generally coupled with a carrier protein), quantification of the injection volume by means of the detection system.
When using our Femtotips, the determined settings can be retained from capillary to capillary. Typical volumes are 0.1 to 0.5 pl for injection into the cytoplasm and 0.01 to 0.05 pl for injection into the nucleus.

Publications:
a) Ansorge, W. and Pepperkok R., (1988): Performance of an automated system for capillary microinjection into living cells. Biochem. Biophys.Meth.16, 283-292 (Calculation by injection with fluorescence markers.)
b) G. Minaschek, J. Bereiter-Hahn, and G. Bertholdt (1989): Quantification of the Volume of Liquid Injected into Cells by Means of Pressure, Experimental Cell Research 183, 434-442 (Explanation of the calculation of the injection volume depending upon pressure and time.)