|
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:
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. |
|
| 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:
- Precipitate the ligated DNA using ethanol or butanol and glycogen
as described in Biotechniques 16, 988.
- 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.
Back to top
Microinjection/Micromanipulation
Consumables
CustomTips
|
|
| Can I specify both the outer diameter and the inner diameter for
CustomTips? |
|
This is only true for CustomTip type II, type holding pipettes.
For the other types: as the raw material has a fixed relation between inner and outer diameter, only
one diameter, e.g. the inner diameter of the capillary can be chosen. The other diameter, in this
example the outer, is fixed.
|
|
| What is the relation between inner and outer diameter? |
|
The relation between outer diameter and inner diameter is 1.25. If you order CustomTips with an
inner diameter of 50 µm the outer diameter will be 62.5 µm (1.25 x 50 µm).
|
|
| What are the dimensions of the CustomTips at the rear end? |
|
The overall length is between 50 and 55 mm, the outer diameter is 1.0 mm. |
|
| Which capillaries can be used for ICSI in cattle? |
|
Please keep in mind that the size of the cell is different in different cattle species. For the
holding side we recommend:
a.) VacuTip, inner diameter 15 µm/ outer diameter 100 µm
b.) CustomTip type II, inner diameter 30 µm/ outer diameter 120 µm
Tip for the injection side:
a) CustomTip number 97/006. This is a sterile TransferTip with an inner diameter of 8 µm.
Femtotips |
|
| How is it possible to distinguish the different types of Femtotips
by the package? |
|
Femtotips are delivered in a silver bag, Femtotips II in a golden.
|
|
| Which tips are the best for injection into the nucleus of MDCK epithelial
cells? |
|
We recommend Femtotip II (930000043). 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.
TransferTip (MDS)
|
|
| Please tell me the diameter of the TransferTips MDS? |
|
The TransferTips MDS have a diameter of 20 µm.
Capillary Safe
|
|
| Which material is used for the Capillary Safe? |
|
It is made of polysterol.
Microloader
|
|
| On which pipette does the Microloader fit? |
|
Eppendorf Pipette Research with variable volume 0.5 - 10 µl (order no. 022471902).
|
|
| Please tell me the material of the Microloader? |
|
The Microloader is made of polypropylene.
|
|
| Are the Microloaders autoclavable? |
|
Yes, the box 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. |
|
| 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? |
|
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.
Back to top
Microinjection/Micromanipulation
Instruments
Micromanipulators
New Manipulator Generation (TransferMan NK2, InjectMan NI2, PatchMan NP2) - General
|
|
| Can a PC be used to control the the New Manipulator Generation (NMG)? |
|
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:
NK2, NI2, NP2”. Both documents are available at our website.
|
|
| What are the main features of the New Manipulator Generation (NMG)? |
|
- 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
- Communication with every lab server
|
|
| Which working distance is necessary for an upright microscope? |
|
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.
InjectMan NI2 |
|
| Can the injection angle be varied to suit different applications
(e.g. DNA injection into the gonads of worms)? |
|
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).
TransferMan NK2 |
|
| I would like to inject into lymphocytes with the TransferMan NK
2. Do you have any recommendations? |
|
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. |
|
| Can the TransferMan NK 2 also be used for injection into adherent
cells? |
|
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.
|
|
| Is the TransferMan NK 2 in combination with a CellTram Air and VacuTips
suitable for the transfer of plant cells? |
|
Yes, this application is possible.
|
|
| How many adapters are required to mount two TransferMan NK 2 on
an inverse microscope? |
|
Only one adapter is necessary.
Microinjectors
CellTram Air/Oil/vario
|
|
| Can I extend the pressure tube of my CellTram? |
|
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: 920002081) in addition to the tube coupling (order
number: 920002421).
The tube coupling can´t be used for older models of CellTram Oil (without quick Valve-Sytem).
|
|
| Can you use different types of oil for filling the CellTram Oil/vario
on the one hand and for covering the cells on the other hand? |
|
Yes, this is possible, as both liquids usually do not come into direct contact.
|
|
| Can you calculate the injection volume with the CellTram Oil or
vario exactly? |
|
No, the mentioned volumes of 0.02 µl (coarse mode) and 0.002 µl (fine mode) are the
moved oil volumes, not the injected volume itself.
|
|
| My CellTram Oil/vario does not react immediately after turning the
dial. Why? |
|
Check the system for air bubbles. If you observe air bubbles, refill the system with
oil. When working with CellTram Oil or CellTram vario with a Quick Valve™, please check the
O-ring (1.5) and exchange it if necessary. Please also check that the grip head is fixed well. |
|
| Is it necessary to use paraffin oil for the CellTram Oil/vario,
or is it also possible to use silicone oil? |
|
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.
|
|
| What is the difference between the Eppendorf CellTram Oil and the
CellTram vario? |
|
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.
|
|
| Which oil can be used for the CellTram Oil/vario? |
|
We recommend the following oil from Sigma. Product-No. M-8410.
|
|
| Can you transfer cell organelles with a diameter of about 10 µm
with the CellTram vario or CellTram Oil? |
|
Yes, that is possible. Please use an appropriate capillary. |
|
| For the holding of cells you recommend the manual injector CellTram
Air in the catalog. Can you also hold cells with the CellTram Oil or vario?
|
|
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.
FemtoJet/FemtoJet express
|
|
| Are all parts that I need for the connection of the FemtoJet/FemtoJet
express to the InjectMan NI 2 included in the standard accessories or are additional parts required? |
|
All necessary cables and tubes are included in the standard accessories. However, the adapter for
the special microscope must be ordered separately.
|
|
| Can FemtoJet/FemtoJet express be controlled externally, e.g. by
a TTL connection? |
|
Yes. The device can be controlled externally via an RS 232 interface.
|
|
| What is the compensation pressure pc? |
|
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. |
|
| Which grip head is included in the delivery package of the FemtoJet/FemtoJet
express? |
|
Grip head number 0. It can be used for capillaries with an outer diameter of 1.0-1.1 mm.
Universal Capillary Holder
|
|
| How do you remove pieces of broken glass capillaries from the capillary
holder? |
|
For this purpose we offer a Service kit (920005888) that contains an appropriate tool. Alternatively
you can also use a thin metal wire.
|
|
The pressure tube connected with the universal capillary holder
920007392 is missing. What do I have to order?
|
|
According to the injectors in use you have to order the following tubes:
Connection to CellTram Air/Oil/vario: 920002081
Connection to Transjector/FemtoJet/FemtoJet express: 920007431
|
|
What is the outer diameter of the universal capillary holder?
|
|
The outer diameter is 4 mm.
|
|
How can the different grip heads be distinguished?
|
|
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.
|
|
| I want to use my own capillaries with Eppendorf microinjectors.
Which capillary grip would fit my tips? |
|
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. 920007414
Capillary grip 1: fits microcapillaries with an outer diameter of 1.2 to 1.3 mm, order no. 910012509
Capillary grip 2: fits microcapillaries with an outer diameter of 1.4 to 1.5 mm, order no. 910012517
Capillary grip 3: fits microcapillaries with an outer diameter of 0.7 to 0.9 mm, order no. 920007406
Transjector 5246
|
|
| Is it possible to connect the Transjector 5246 to the manipulators
of the new generation? |
|
Yes, this is possible. For the combination of Transjector 5246 and InjectMan NI 2 you must use
the following interface cable (920005853).
Back to top
Microinjection - General
|
|
How do I convert from hPa to psi?
|
|
1 hPa = 0.0145 psi
1 psi = 68.95 hPa
|
|
| What parameters are decisive for the injection volume, and how can
I determine these? |
|
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:
- 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.
- 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.
- 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.)
|
|
| What can you recommend with regard to sample preparation of the
injection solution for microinjection? |
|
- 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.
|
|
| What should be taken into account when injecting proteins? |
|
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.
|
|
| Do you have a publication which explains how to determine the diameter
of microinjection capillaries and also 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 Hotline (application-hotline@eppendorf.de).
Back to top
Microinjection – adherent cells
|
|
| Is any literature available on the subject of microinjection of
adherent cells using Eppendorf devices? |
|
Yes, there are many publications involving Eppendorf devices including:
- Habedanck R., Stierhof Y.D., Wilkinson C.J., Nigg E.A. (2005) The Polo kinase Plk4 functions
in centriole duplication. Nat. Cell Biol. 7: 1140 - 1146
- Stephens D.J., Pepperkok R. (2004) Differential effects of a GTP-restricted mutant of Sar1p
on segregation of cargo during export from the endoplasmic reticulum. J. Cell Sci. 17: 3635 - 3644
- Uchida A., Brown A. (2004) Arrival, reversal, and departure of neurofilaments at the tips of
growing axons. Mol Biol Cell 15: 4215 - 4225
- Watson P., Forster R., Palmer K.J., Pepperkok R.; Stephens D.J. (2005) Coupling of ER exit to
microtubules through direct interaction of COPII with dynactin. Nat. Cell Biol. 7: 48 - 55
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| Can you recommend any parameters for the injection of DNA into adherent
cells? |
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Parameters for the injection of DNA into mammalian cells using Femtotip or Femtotip II:
pc = 30 to 300 hPa
pi = 50 to 500 hPa
ti = 0.3 to 1.5 s
The parameters must be optimized according to each experiment. If you are using Femtotips, please
mount the universal capillary holder at an angle of 45°.
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| What is the typical injection volume? |
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The injection volume is typically ~ 10% of the cell volume.
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| Can you supply us with literature about microinjection of DNA or
proteins into adherent cells? |
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Our Application No. 8 contains interesting hints about the preparation of samples for microinjection.
www.eppendorf.com
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| What guidelines are available for the optimal concentration of protein
or plasmid in the injection capillary? |
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As a rule, 20-200 ng/µl DNA and 1–5 mg/ml protein are injected. With an antibody solution,
the concentration should be 10 to 50 times higher than that used in the in vitro test system (e.g.
Southern blot).
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|
| I inject 0.5 % FITC-labeled dextran into MEL (mouse erythroleukemic)
cells. Although I centrifuge the injection solution before loading the capillary and make sure that
the solution doesn't dry up in the capillary, I am unable to perform injection. What can I do? |
|
- Is liquid coming out of the capillary? Please press the "Clean" button on FemtoJet/FemtoJet
express. If no liquid is coming out of the capillary, the capillary holder or the Femtotip adapter
may be clogged. Please check.
- It is possible that splinters of glass from a capillary used in a previous experiment are blocking
the capillary holder. These splinters can be removed using a long, thin metal needle (contained
in the Service Kit).
- Is the injection time long enough? If the injection time is too short, the FemtoJet/FemtoJet
express may reduce the pressure to the compensation pressure before the injection level has been
reached (before the cells are permeated). In this case, your injection would be performed using
the compensation pressure only.
- If the injection solution has a high viscosity, you may have to work with higher pressures.
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| Can I use Trypan Blue for the demonstration of a microinjection
procedure or should I use fluorescent dyes? |
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If you are using Trypan Blue, the capillaries may become clogged easily. We therefore recommend
the fluorescent dye FITC Dextran (e.g. from Sigma)
Procedure:
- Make a stock solution 10 mg/ml and filter with a 0.2 µm syringe filter
- Concentration: 0.5-1.0 mg/ml in microinjection buffer together with the sample.
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| Which dextran is most suitable for microinjection? |
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All dextrans can be used irrespective of the molecular weight. For a special application, namely
compartment-specific injections, we use dextran with a molecular weight in excess of 60 kDA, as it
cannot penetrate the core barrel.
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Microinjection – suspension cells
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| I work with your TransferMan NK 2 and two CellTram Air at my ICSI
work place. I can hold the cells perfectly, unfortunately I am unable to transfer or inject them. |
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CellTram Air injectors are perfect for holding cells. The advantage of the CellTram vario in comparison
to the CellTram Air is a more precise movement control because of the oil in the system. We therefore
recommend using the CellTram vario for the injection side.
A list of the components of an ICSI work place can be found in our main catalogue or at our homepage.
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Microinjection – small organisms
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| Which Eppendorf devices do you recommend for the injection into
insect embryos? |
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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.
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| Which injection pressure do I have to select for microinjection
into fish embryos? |
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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.
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| Do you know of any publications on injections into Caenorhabditis? |
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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.
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Microinjection – plant cells
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| Can you recommend any literature about microinjection into plant
cells? |
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- Schnorf, M. et al., An improved approach for transformation of plant cells by microinjection:
molecular and genetic analysis, Transgenic Research 1, 23-30 (1991)
- Brücker et al., Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient
mutants of the moss Ceratodon purpureus, Planta 210, 529-535 (2000)
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| What is important for the microinjection into plant cells? |
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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).
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| What equipment is required to inject fluorescent dyes into plant
cells and to then aspirate the entire content of the cell? |
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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.
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Microdissection
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| How does your microdissection device work? |
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Microdissection of histological preparations is undertaken with the MicroDissector and a Transferman
NK 2. An ultrafine metal chisel is operated at high frequency and low amplitude with the help of
a piezo-stepper that vibrates in the ultrasonic range. This means that single cells fixed on a slide
can be isolated. The isolated cells are aspirated with a special pipette and transferred to a Safe-Lock
tube. Afterwards, all the usual downstream applications such as RT-PCR or quantitative mRNA expression
analysis can be carried out.
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| Which manipulator do you recommend for microdissection? |
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We recommend the TransferMan NK 2.
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| Can the Eppendorf MicroDissector also be mounted on manual manipulators? |
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Yes, both the cutting tool and the electronic pipette can be mounted on manual manipulators if
they have a tool holder for tools of about 4 mm in diameter.
|
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| Could you tell me the dimensions and the volume of your Filtertips
MDS for microdissection? |
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Filtertips MDS have an inner diameter of 150 µm and a volume of 30 µl.
|
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| Can you recommend an alternative to xylene as a coating agent for
your cryosections? |
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Yes. Stephens Scientific citrus-based clearant, Catalog no. 8301 from Richard-Allen
Scientific or Histoclear from National Diagnostics, Atlanta
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| Can the Eppendorf MicroDissector also be used for cutting chromosomes? |
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For this application it is sufficient to use a precise manipulator, e.g. the TransferMan
NK 2. The chromosomes are so tiny that they fragment even when touched. They stick to the tip because
of electrostatical power. Just break the tip into a tube. Your sample can then be extracted by standard
kits.
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| How is the tip of the MicroChisel shaped? |
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It is a uniform tip.
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