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. This may affect the electrical conditions in the electroporation cuvette and can lead to a lower transformation rate. Therefore the conductivity of the ligation preparation should be reduced by standard methods as for instance:
Precipitate the ligated DNA by adding ethanol or butanol and glycogen as described in Biotechniques 16, 988. Dissolve the precipitation in an appropriate volume of water.
Is it necessary to use classic competent cells for the electroporation of bacteria?
No, "classic" competent cells are needed only for transformation according to the CaCl2 method. For electroporation so-called "electrocompetent" cells are required. In contrast to classic competence, the permeability of the cell membrane is not increased, but the bacterial preparation should have a low conductivity . This preparation involves harvesting at a particular cell density, washing steps with water or low-salt buffers and setting a cell concentration suitable for electroporation. In the case of relatively large preparations, glycerin is also added to allow the cells to be deep-frozen (in aliquots).
What kind of electroporation cuvettes are compatible with the Eporator and in the Multiporator?
Every standard electroporation cuvette using a cap with a square-shaped edge are compatible with the Eppendorf electroporators. E.g. the electroporation cuvettes of following brands fit: Cellprojects, Bio-Rad, Peqlab, Chemglass ... (Status 2018). Cuvettes using a cap with a round-shaped edge do not fit (e.g. BTX)
Can the time constant on the Eppendorf Eporator be adjusted?
The time constant of the Eporator has a fixed setting of 5 ms which corresponds to the optimal pulse length for bacteria. The actual time constant given (depending also from the electroporation conditions within the cuvette) is displayed right after the pulse.
Can the electroporation protocols for bacteria and yeasts of the Multiporator also be used for the Eppendorf Eporator ?
Yes, protocols using the bacterial module of the Multiporator (Mode- prokaryotes “O“) can be transferred to the Eppendorf Eporator
Could you please tell me the order number for the Femtotip adapter?
Ordering number for the Femtotip adapter is 5176 208.009.
How is it possible to distinguish the different types of Femtotips by the package?
The Femtotips and Femtotip II can be distinguished by the color of their protective plastic cap:
Femtotips®: colorless protective cap
Femtotip II: light yellow protective cap
Which tips are the best for injection into the nucleus of MDCK epithelial cells?
We recommend Femtotip II (5242 957.000). They have an inner diameter of 0.5 µm and an outer diameter of 0.7 µm, which is very practical for injection into these cells.
Which material is used for the Capillary Safe?
It is made of polysterol.
Which Material is used for the custom tips?
Raw material is borosilicate.
Please tell me the material of the Microloader?
The Microloader is made of polypropylene.
Are the Microloader tips autoclavable?
Yes, the box including the tips can be autoclaved up to five times.
Are the Microloaders free of RNase? How can you clean them?
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.
On which pipette does the Microloader fit?
Following pipettes are suitable for the microloaders: The pipette Eppendorf Research plus with variable volume 0.5 - 10 µL (order no. 3123 000.020) and the pipette Eppendorf Reference 2, either with variable volume 0.5-10 µL (order no. 4924 000.029) and 2.0-20 µL (order no. 4924 000.037).
Please tell me the length of the Microloader tip?
Length approx. 100 mm in total, from conus of pulled tip approx. 65 mm.
Please tell me the outer and inner diameter of the Microloader on the tip?
The thin pulled tip of the Microloader has an outer diameter smaller than 0.45 mm
How do you remove pieces of broken glass capillaries from the capillary holder?
For this purpose we included a Cleaning stylet in the bag of the capillary holder 4, which is included in the delivery scope of the microinjector. Alternatively you can also use a long thin metal wire. The capilary holder 4 (incl. 1x Grip head set 4 size 0; 1x Adapter for Femtotip, 1x removal tool, 1x Cleaning stylet) can be seperately ordered (5196 081.005).
I used before a capillary which was named 'Polar body biopsy Tip MML' (Order No.5175 210.000). I can't find it anymore on your homepage. You don't have it anymore?
All our microcapillaries got new order numbers (April 2018). And for few capillaries we changed their names to simplfy them. The 'Polar body biopsy Tip MML' (Order No.5175 210.000) is now called biopsy Tip I (Order No. 5195 000.052)
What are the main features of the Eppendorf Manipulators?
* High speed for efficient penetration of rigid structures (Vmax 10,000 µm/sec)
* Motors with high resolution for smooth step-free motions
* Intuitive operation concept and easy and ergonomic handling via one joystick controlling all motor movements
* Smart functions like position storage, Home fucnction, step injection etc, allowing to support a smooth and fast workflow
+ simple and individual adaption of the motor speed
Can the TransferMan also be used for injection into adherent cells?
In principle it is also possible to use the TransferMan for injection into adherent cells. However, our InjectMan is much more suitable for this application. The unique electronic coupling of the InjectMan 4 with the microinjector FemtoJet makes microinjection into adherent cells very quick and easy. The TransferMan with its proportional joystick is specialized for the manipulation of mammalien oocytes or early embryos.
Is it possible to apply the same parameters from other piezo devices e.g. PMM onto the PiezoXpert and get the same effects?
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.
What does Intensity stand for?
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.
What does Speed stand for?
As speed we define the number of pulses per second.
Why do I have to use a distance plate when I mount the Actuator of the PiezoXpert onto a
The distance plate secures that the Piezo element at the actuator does not get in direct contact with any part of the manipulator which would have an impact on the piezo-impulses transferred to the microcapillary. The distance plate is not necessary when you want to mount the PiezoXpert onto the TransferMan 4.
TransferMan NK 2?
Why is FluorinertTM FC-77/ FC-770/FC-40 or mercury used for e.g. mouse ICSI?
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.
How is mercury filled into the capillary? How much of mercury should be used?
Filling of mercury into the capillary can be done by backfilling using some special syringe e.g. Hamilton syringe. Usually 1-4 µL of mercury is sufficient. Note that mercury is toxic, please backfill the capillary under a fume hood.
When I triggered the pulses, no visible vibration of the capillary was observed. Is that normal?
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.
I have tried high intensity but still failed to penetrate the cell. There seems to be no impulses being transmitted to the capillary. What should I do?
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.
I sometimes fail to perform penetration through zona pellucida and plasma membrane separately. The capillary advanced directly into the oocyte. What should I do?
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.