Frequently Asked Questions
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1. The Electroporator 2510 qand 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.
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.
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.
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 Helix fusion chamber can be filled with 250 µl of sample.
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).
The chamber should be cleaned with distilled water before the cells have dried out. Heavier contamination can also be removed using a soft toothbrush or a cleaning agent (e.g. Edisonite Super, order no. 60210, VWR). It is possible to sterilize the chamber by treating it with ethanol (70 %, non-denatured), but the Micro fusion chamber is an open system and normally serves only to optimize parameters.
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, all Multiporators can be upgraded with the respective upgrade kits. Please note that the module has to be installed by a service technician.
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.
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.
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. .
The maximum number is 99, with an interval of 60 seconds in each case.
"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"
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.
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.
No, this is not recommended. 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.
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.
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.
The buffer components are listed in the relevant application manual for electroporation/electrofusion.
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.
The cuvette stand for electroporation cuvettes is made of polypropylene and can be autoclaved at 121 °C.
Yes, it is possible.
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.
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
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.
We recommend TransferMan NK 2 or InjectMan NI 2, FemtoJet express and a footswitch for the FemtoJet express. For objects with a strong shell (Chitin) a Piezo Drill may be necessary.
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.
A PC can easily control all Eppendorf manipulators of the new generation. A detailed description can be found in the operating manual and in the “Supplement: Change in software from 1.06 upwards: NK 2, NI 2, NP 2”. Both documents are available at our website.
- 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
The minimum working distance is approx. 5 mm. In this case the capillary has to be mounted very flat, almost parallel to the table. In general, inverted microscopes are the better choice for micromanipulation experiments because of their large working distance of more than 20 mm.
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).
Yes, this application is possible.
Only one adapter is necessary.
Microinjection into lymphocytes is a challenge because of their small diameter of approx. 5 µm. This cell type can be adherent or in suspension. In the case of adherent cell culture they can be injected by users with some experience. If you work with lymphocytes in suspension cell culture, you also have to use capillaries with a very small diameter (e.g. CustomTips II) for holding the lymphocytes.
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.
Since a very high pressure is required for injection into some plant cells, we recommend using the manual CellTram Oil Microinjector in combination with a micromanipulator (e.g. TransferMan NK 2), which enables material to be extracted out of the cell. If suspension cells are used, a second micromanipulator is necessary to fix the 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).
"1. Schnorf, M. et al., An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis, Transgenic Research 1, 23-30 (1991)
2. Brücker et al., Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus, Planta 210, 529-535 (2000) "
"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, two pressure tubes can be connected with the help of a tube coupling. Please note that you also have to order a pressure tube (order number: 5176 114.004) in addition to the tube coupling (order number: 5176 220.009). The tube coupling can´t be used for older models of CellTram Oil (without quick Valve-Sytem)."
Yes, this is possible, as both liquids usually do not come into direct contact.
You can use paraffin or silicone oil, depending upon the viscosity required for your application. Please clean the device very carefully when changing the oil.
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.
We recommend the following oil from Sigma. Product-No. M-8410.
Yes, that is possible. Please use an appropriate capillary.
As the devices differ in their maximum pressure, we recommend the CellTram Air for the holding of cells and CellTram Oil or vario for the transfer of cells (max. pressure of the CellTram Air 2,900 hPa/ max. pressure of the CellTram Oil/vario 20,000 hPa). For special applications (e.g., biopsies) we also recommend the CellTram Oil or vario also for holding, as a stronger vacuum is necessary for fixing the cells properly.
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 grip heads have different numbers of notches. The grip head 0 has no notch at all; the grip head 3 has, for example, three notches
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.
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.)
"- 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 (from the rear). Use it only once. The liquid should be extracted from the top of the tube. Make sure that no gas bubbles are in the glass capillary.
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 danger of the injection solution drying in the capillary, thus blocking the Femtotips. Please find more detailed information in our Applications No. 8 "Sample preparation for microinjection", which we are happy to send you upon request."
"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."
It is sometimes difficult to inject protein solutions, as any contamination can block the capillary. Therefore, the solutions must be prepared very carefully (e.g. cleaned by centrifugation columns or ultracentrifugation). Before loading the Femtotip, the solution should always be centrifuged for at least 10 minutes at the highest speed
The Microloaders are free of RNase because of the heat during production. This was confirmed by a test of the PCR-clean procedure. Because of the small tip diameter, a further cleaning process is not recommended.
Length approx. 100 mm in total, from conus of pulled tip approx. 65 mm.
The thin pulled tip of the Microloader has an outer diameter of approx. 0.25 mm and an inner diameter of approx. 0,20 mm
Eppendorf Pipette Research with variable volume 0.5 - 10 µl (order no. 3111 000.122) andthe Eppendorf pipette Refernce with variable volume 0.5-10µl (order no.4910 000.018) and 2.0-20µl (4910 000.026).
All necessary cables and tubes are included in the standard accessories. However, the adapter for the special microscope must be ordered separately.
Yes. The device can be controlled externally via an RS 232 interface.
The compensation pressure pc ensures that no culture medium flows into the capillary during microinjection experiments. Capillary forces would make liquid flow out of the cell culture dish into the injection capillary and thus dilute the injection material. To prevent this, a permanent compensation pressure pc is set. This should be selected so that there is a permanent slight flow-out of liquid from the injection capillary. The individual pressure level can be determined in a preliminary test.
Grip head number 0. It can be used for capillaries with an outer diameter of 1,0-1,1 mm.
Maybe not. This is because the design of each piezo device is different e.g. distance of piezo element and the capillary, installation of capillary holder and size of piezo element. Usually, parameters with lower strength of PiezoXpert are used compared to other piezo devices due to its optimized design.
"Intensity stands for the level of pulse strength or impact. It can be set on a scale from 1-50 for program setting and 1-86 for the Clean function. The max. intensity of 86 equals 732 V."
The spacer plate functions as a “spacer” so that the actuator is not in direct contact with the micromanipulator. If it was, the piezo impulses could not be transferred directly and reproducibly onto the attached microcapillary.
"The use of liquid with high density is to give extra “weight” on the capillary that gives higher impact of piezo impulses for a successful perforation."
"Filling of mercury into the capillary can be done by backfilling using some special syringe e.g. Hamilton syringe. Usually 3-4 μL of mercury is sufficient. Note that mercury is toxic, please backfill the capillary under a fume hood."
Yes, it is normal. The design of the actuator element is optimized (directly adjacent to the capillary and rigid installation) in order to give efficient impulses directly to the target cell. This way, vibrations that could cause cell lysis are reduced. However, you can hear the impulses and also get a visual feedback via the illuminated rings around the buttons directly on the device.
"Make sure the capillary is inserted deep enough into the grip head. The capillary should be inserted until it touches the stopper inside the holder in order to allow direct transmission of the impulses onto the capillary."
"Improve the control of the micromanipulator. The use of an electronic micromanipulator where speed can be reduced helps. Reduce the strength of the piezo impulses by decreasing the settings for int. and speed."