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Eppendorf Cooler and Mixer F.A.Q and Troubleshooting

IsoTherm Benchtop Cooler

The Eppendorf IsoTherm Systems are designed to cool samples effectively and consistently over many hours at 0°C or at –21°C. The White IsoTherm System is designed for 0°C and the Blue IsoTherm System is designed for –21°C. The Systems consist of the following components: the IsoPack (cold pack), the IsoRack (transparent storage rack), and the IsoSafe (insulated transport box).

How long do IsoPacks maintain their temperature?

White IsoTherm System: IsoPacks that are seated in IsoRacks and sealed in the insulating IsoSafe can maintain a temperature of 0°C up to six hours. If the IsoPacks are contained in an opened IsoSafe, then a temperature of 0°C can be maintained for approximately 4 hours. If IsoPacks/IsoRacks are not placed in an IsoSafe, then 0°C can be maintained for 1.5 to 3 hours. Blue IsoTherm System: IsoPacks that are seated in IsoRacks and sealed in the insulating IsoSafe can maintain a temperature of –21°C for over 3 hours. If the IsoPacks are contained in an opened IsoSafe, then a temperature of –21°C can be maintained for 1 to 2 hours. If IsoPacks/IsoRacks are not placed in an IsoSafe, then –21°C can be maintained for 0.5 to 1 hour.

At what temperatures can the IsoTherm Rack be stored?

The IsoTherm Rack can be stored at temperatures as low as –200°C.

What are IsoRacks made of?

IsoRacks are made of polyethylene and filled with a non-toxic liquid.

What are the IsoPack’s dimensions? What are the IsoRack’s dimensions?
DescriptionDimensions (W x D x H)
IsoPack for 0.5 ml tubes4.7 x 3.0 x 0.9 in./12 x 7.7 x 2.3 cm
IsoRack for 0.5 ml tubes, with lid5.0 x 3.4 x 1.9 in./12.7 x 8.6 x 4.9 cm
IsoPack for 1.5/2.0 ml tubes4.8 x 3.1 x 1.5 in./12.2 x 8 x 3.9 cm
IsoRack for 1.5 and 2.0 ml tubes, with lid5.0 x 3.4 x 2.5 in./12.7 x 8.6 x 6.4 cm
Are IsoPacks resistant to sodium hypochloride (NaOCl)?

Sodium Hypochloride (NaOCl) will attack the aluminum foil on both the blue and the white IsoPacks to some extent. Over a period of eight hours or more, this may lead to damage. The IsoPack’s polyethylene housing is chemically resistant to NaOCl. Even if a leak occurs, the liquid in the IsoPack is non-toxic.

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Thermomixer R

Why can’t I set the temperature higher than 70°C with the block for microplates (Brinkmann Catalog # 022670565)?

Many microplates are made of polystyrene, which becomes soft at 80°C. As a safety precaution, the Eppendorf Thermomixer R will not exceed temperatures of 70°C when using the block for microplates. If you are using polypropylene or Teflon® plates that are rated to 99°C, you may disable this safety feature.

Disclaimer 
To avoid risk of personal injury and damage to the Thermomixer, please be sure that when disabling this feature, only microplates and deep-well plates rated to 99°C are incubated.

In order to disable this safety feature, you must deactivate the block recognition system. Please follow the procedure outlined below.

  1. Turn power on and set the speed to 500 rpm
  2. Turn the power off
  3. Turn the power on while holding the Temperature down arrow key; the display will indicate “service ON”
  4. Scroll through the menu by pressing the Temperature up arrow key until the display indicates “Block ID”
  5. To turn the block recognition off press the Mix up arrow key. To turn the block recognition on, press the Start/Stop button.
Do I need to fill the thermal block holes with water?

No. Eppendorf Thermomixer R takes advantage of Peltier technology, therefore cooling or heating fluid is not necessary. Filling the thermal block with water will damage the Thermomixer.

I have an older Eppendorf Thermomixer Model 5436. Can I use the Model 5436 thermal block with the Thermomixer R?

No. The model 5436 thermal block is not compatible with the Thermomixer R.

I need to cool my samples to 4°C. Can I run the Thermomixer R in a cold room to maintain 4°C?

Yes and no. The Eppendorf Thermomixer can be operated in a cold room, however, precise temperature control at 4°C is not guaranteed.

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Laboratory Pipette F.A.Q and Troubleshooting

Pipettes:

How do I calibrate my pipette?

The Brinkmann Standard Operating Procedures (SOP) Manual for Pipettes is a reference guide for the correct way to gravimetrically test all Eppendorf pipettes. Information included in the SOP manual details the setup of the test station, the proper testing procedure and specification reference charts for all Eppendorf pipettes

Is there a sterile version of the Reagent Reservoir?

No. Reagent Reservoirs are in packs of ten with a lid. They are autoclavable, but not sterile (catalog no.: 022265806)

What is the Reagent Reservoir made of?

Both the Reagent Reservoir and the lid are made of polypropylene.

Is the Eppendorf Research pro Electronic Pipette autoclavable?

The lower portion of the pipette can be removed and autoclaved.

Are the Maxipettor and the accompanying Maxitips resistant to 40% iron trichloride?

Yes.

Are Eppendorf pipettes free of nucleic acids?

We are unable to guarantee this 100 %. Although all safety and hygienic precautions are observed during production, no specific action is undertaken to prevent the infiltration of human nucleic acid. To ensure sterility and to protect your pipettes from contamination, we recommend using the Eppendorf Positive-Displacement Pipettes or Filtertips.

What can you recommend regarding the handling of viscous liquids?

Our recommendation:
1) Positive-Displacement Pipette with Mastertips (positive-displacement principle)
2) Reverse pipetting
3) Repeater Plus

Is the Eppendorf Research Series 2100 pipette autoclavable?

The complete lower part of pipette is autoclavable. Pull off / unscrew the ejection sleeve and disassemble the lower part using the tool provided. The pipette can also be decontaminated using UV light.

What is the best way to pipette chloroform?

The most suitable tool is the Eppendorf Positive-Displacement Pipette (with Mastertips) or Repeater Plus (with Combitips).

Which DIN 12650 standard is valid for single-channel, piston-stroke pipettes?

The DIN 12650 standard of 1978-83. Drafts are available for 1998 (DIN 12650-1, -2, -6), although these have yet to be authorized.


Pipette Tips

Genuine Eppendorf Pipette Tips are designed to ensure certified performance of all Eppendorf Pipettes. A wide variety of Eppendorf Pipette Tip sizes, styles, and packaging options offers convenient choices for the best performing tips to meet your application needs. Eppendorf Combitip Plus Tips are designed for use with the original Eppendorf Repeater® Pipette, the Eppendorf Repeater Pro, and the EDOS 5222 Dispensing System. The positive displacement tip design ensures accuracy, even when handling viscous or high vapor pressure solutions.

Are the polypropylene Eppendorf Pipette Tips UV-resistant?

Yes. Therefore it is completely safe to use them on a sterile bench.

Do Eppendorf Pipette Tips contain a softening agent?

No.

What is the PerfectPure C-18 Tip?

The Eppendorf PerfectPure C-18 Tip uses hydrophobic interactions to reversibly bind, purify, and concentrate peptides and small proteins. Porous silica particles functionalized with C-18 beads (commonly used for reversed-phase chromatography) are fixed at the outlet of the tip.

What is the compatibility of the C-18 tips and Eppendorf pipettes with the chemicals using in the above protein analysis?

Eppendorf PerfectPure C-18 Tips are compatible with all materials listed in the manual.

How quickly can I desalt and concentrate peptide or protein samples with the C-18 tips?

The bind, wash, elute protocol takes less than 2 minutes to complete.

What are the specifications of the C18 in the C-18 tips (material, pore size, etc)?

The silica particles used in the Eppendorf PerfectPure C-18 Tips is functionalized with carbon-18 chains commonly used in reversed phase chromatography. The particles range in size from 32–63 microns, and have 100 angstrom pores.

What are the sample elution volumes from the tip?

Volumes as small as 0.5 microliters have been used successfully, however evaporation is a problem with this small volume of a very volatile liquid and usually larger volumes (1–3 µl) provide more reproducible and reliable results. Larger volumes (up to 10 µl) have been verified as well.

What maximum volume can be used with the tip?

Up to 50 µl of analyte has been bound with success. If a larger volume is required, increase the number of binding steps and ensure that all the liquid passes through the plug of the tip at least twice.

How do I process sample volumes larger than 10 µl through a C-18 tips?

See above question. Ensure that all the liquid passes through the plug at least twice —increase the number of binding cycles if necessary.

What is the minimum amount of sample I can recover from the C-18 tips?

This number is dependent on a variety of factors, most importantly the sensitivity of the MS machine used. In house we have detected down to femtomole quantities which is at the limit of our machine. A beta-tester showed recoveries in the attomole range, however this data is not available for publication.

Are the tips compatible with robotic / automated sample preparation systems?

At present we are not able to cover all robots for sample preparation (MALDI Spotters). In spring 2004 the application for Eppendorfs epMotion will be available. For other workstations Eppendorfs OEM department is in charge to find solutions (fixing the C-18material in fitting tips).

What pipettes (manufacturer, volume, single or multi channel) are compatible with the C-18 tips?

Eppendorf PerfectPure C-18 tips are compatible with all standard p10 (10 µl) pipettors (Eppendorf, Rainin, Gilson, etc…). The tips have been tested using manual single and 8- channel, as well as electronic single and 8-channel Eppendorf ResearchPro pipettes.

When should I consider using the step gradient elution protocol?

Step gradient elution can be used to perform a simplification of mass spectra by partial fractionation of complex peptide mixtures. This leads to a minimization of peak supression of larger peptides. One can also do a fractionation of peptides according to their hydrophobicity.

Performing a step gradient elution with the C-18 tips, how can I eliminate carry over from one step in a gradient to the next?

Cross contamination can be avoided by performing additional washing steps in betweenthe elutions.

I would like to separate a peptide from a protein. Is it possible to fractionate using the C-18 tips?

Due to the pore size in the silica particles, the molecular weight cut-off for efficient binding is around 15,000 Daltons. And the C-18 functionalization is a strong hydrophobic molecule used to bind smaller peptide fragments, such as those created in a trypsin digest. If the protein you are trying to separate is significantly larger than 15,000 Daltons, you could try using the tip to bind and elute the peptides and the protein would be left in the “flow-through”.

During the elution step, an air bubble was introduced into the tip. Will this decrease the recovery of peptides?

This should not have any effect on your recovery, the significant proportion of hydrophobic solvent in the elution solution passes easily through the tip. However, occasionally in the early stages of the protocol, an air bubble will cause significant backpressure that cannot be overcome by the pipet. If this happens, immediately elute the sample (using the indirect elution solution (no crystallization matrix), dilute the solvent to ~5-10% and start the protocol again from the beginning using a clean tip.

With smaller elution volumes (0.5–1µl) you should also be careful that the bubble is not so big that the surface tension between the pipet and your eluate is broken and you form a bead of eluate on one-side of the pipet above the plug.

I have detergent in my sample; will I be able to enrich my peptide digest with the C-18 tips?

The manual recommends that the analyte should be diluted so that the final concentration of a detergent is at a low enough level to prevent foaming during the pipetting protocol. (<0.1% SDS, <1% Triton, <0.5% Tween).

I don’t want to analyze my mass spec product immediately after elution from the C-18 tips can I store the eluted product safely for future analysis?

This is not recommended. The samples can be stored before they have been desalted, however once desalted, the samples are in a very volatile elution buffer which facilitates evaporation/crystallization on the target. The small elution volumes will readily evaporate, and we have not had much success in-house trying to resuspend samples where the eluate has evaporated. Eppendorf therefore recommends to perform the C-18 Tippurification procedure immediately before the mass spec analysis.

Not all of my peptides were recovered when using the C-18 tips. What should I do?

First try increasing the number of binding and elution pipetting cycles. Second, try one of the optional elution prototcols which implement n-proponal, a significantly more hydrophobic solvent which has been shown to increase recovery profiles in some applications.

I’m performing electrospray / nanospray mass spec on my eluted sample from the C-18 tips. What is the appropriate solution to elute my sample with?

Desalting and concentration are most commonly needed for matrix-assisted laser desorption ionization (MALDI). Because the salts must be removed to aid in crystallization of the sample on the target. Electrospray/nanospray is commonly used to interfaces liquid chromatographic (LC) separation techniques with mass spec analysis. The LC step effectively desalts the sample, and eluted fractions from the LC (in similar solvents) are infused directly into the electrospray/nanospray needle for MS analysis.

How many times do I have to cycle the sample through the C-18 tips to achieve binding to the resin?

This is sample dependant. However, we have found that for most applications 5-10 cycles are plenty. If you feel that you are getting a low recovery, try increasing this number along with the number of elution cycles.

How can I improve salt removal with the C-18 tips?

Increase the number of wash cycles. However, we saw no significant difference in MS spectra for samples spiked with 2M urea which were desalted using 2 wash cycles and 10 wash cycles.

How can I improve binding of hydrophilic peptides and proteins to the C-18 tips?

Try the addition of chaotropic salts and/or increasing of binding steps (20 to 30 x).

Can the C-18 tips be reused?

This is not recommended.

After elution from the C-18 tips my spots won’t dry. Want can I do?

You can try adding more crystallization matrix (Direct Elution Solution) to the spot.

Can I use higher concentration of ACN (> 50%) to pre-wet or elute my sample from the C-18 tips?

Yes. Up to 95% ACN has been tested in-house. You can also use a higher concentration of TFA or Formic Acid to ion-pair your sample (up to 2% has been tried in house).

Is a protocol for elution using optional elution buffer (D2 or D3) available?

It is the same protocol as for the standard elution buffers. See the manual for details.

Does slower aspiration and discarding of the sample solution cause better recovery? Or which level of the aspiration speed is recommended for binding peptides to matrix when we use Eppendorf Research Pro?

With manual pipetting no significant differences were found. With the electronic pipets please refer to the Electronic Research Pro Application Note on our web-site. Although no significant differences were seen in the spectra, %-recovery assays suggest that as expected, slower aspiration speeds achieve higher levels of binding than the faster aspiration speed. The speed you use should be optimized for your analyte/concentration.

Why are peptides >15,000 Da not bound to the C-18 matrix?

This is due to the size of the pores in our C-18 silica particles. We found that this pore size gave optimal binding characteristics for its intended application: peptides, especially those from protein digests. In the future, tips will be developed which are specifically designed for larger molecular weight proteins.


Eppendorf Combitip Plus Tips

What are Combitips Plus Tips made of?

The cylinder is made of polypropylene (PP) and the piston is made of polyethylene (PE).

What certificates are available for Combitips Plus Tips?

A certificate of quality for Combitip Plus Tips is available. Since Combitip Plus tips are also supplied as Eppendorf Biopur, a batch-specific Eppendorf Biopur quality certificate is also available. Eppendorf Biopur products are guaranteed to be sterile and free of pyrogen, RNase, DNA, and ATP.

Which adapter is required for the 25 ml Combitip Plus tip?

The new Repeater Plus Pipette with the blue lever requires the blue adapter (nonsterile adapter, each: catalog number 022266993; Biopur adapter, pkg. of 7: catalog number 022496158).

Is the Eppendorf Combitip Plus Tip Mounting Rack autoclavable?

Yes, up to 50 times.

What is the Combitip Plus Tip Mounting Rack made of?

Polycarbonate, with silicon “feet.”


Dispensers

Does EDOS 5222 recognize Combitips plus?

All EDOS 5222 models recognize the new Combitips plus. Upwards of software version 3.1 and device number 927, the new 25 ml Combitip plus is also recognized.

Can the automatic tip ejector of EDOS 5222 be controlled via a computer link-up?

Yes. This is possible in combination with “REDOS”, the EDOS programming package.

Is it possible for the space between the EDOS control unit and the dispensing grip (hand key) to be longer than 1.5 m (= the same length as a cable)?

The maximum length of the extension cable is 1.5 m. A longer cable would adversely affect data transfer between the EDOS control unit and the dispensing grip.

Is the 25 ml Combitip plus recognized by EDOS 5222?

The 25 ml Combitip is recognized by EDOS 5222 upwards of device number 831 and software version 3.02.

What is the warranty period for the Repeater® Plus?

The warranty period for the Repeater Plus is two years from the day of purchase.

What is the warranty period for the Repeater pro?

The warranty period for the Repeater pro is one year from the day of purchase.

Which tips are suitable for the 8-channel pipetting adapter for 5–50 µl of the EDOS 5222?

The 200 µl tips should be used for this purpose.


Easypet Pipetting Aid

Is the Easypet filter hydrophobic?

Yes. The Easypet filter is made of PTFE (polytetrafluorethylene) and is hydrophobic. It can be autoclaved five times. (0.45 µm sterile hydrophobic filter, set of ten, Catalog no.: 022231006)

Is the Easypet housing resistant to UV irradiation?

Yes. However, high UV doses may lead to slight discoloration of the material.

Can membrane filters with a pore width other than 0.45 µm be used?

Yes, pore widths of 0.3 µm and 0.2 µm may also be used. This has only a minimal effect on aspirating and dispensing speeds. When purchasing, please note the shape of the filter connections; double-sided Male Luer Lock.

Is it possible to continue working with the power unit when the battery is flat?

Yes. The device is recharged during the pipetting process.

How long is the operating life of the battery?

A fully charged battery can be used for approximately 7 hours.

How long does it take to recharge a completely flat battery?

12 hours.

Can the battery be recharged at any time?

In everyday operation, the battery should not be recharged until the charging-control display has lit up. The remaining charge is then approximately 15%

Can primary-cell batteries also be used to power the device?

No. There are no primary-cell batteries with the same dimensions of the three prescribed batteries. These can be ordered direct through Brinkmann using catalog number 022236016, three batteries must be ordered.

Is Easypet autoclavable?

The nose cone and the adapters are autoclavable. The membrane filter may be autoclaved a maximum of five times.

Pipette on the Easypet is leaking. What is the reason for this?

Please ensure that the pipette has been inserted far enough into Easypet. Maybe the adapter or the pipette is damaged.

What is a possible reason for reduced suction capacity?

Reduced suction capacity may be caused by a wetted membrane filter or a discharged battery.

The pipette does not fit tightly onto Easypet. What might be the reason for this?

Please check to see whether the adapter is damaged.

Which batteries are used in Easypet?

Nickel-cadmium batteries are used in Easypet.

How long is the life of the batteries?

If the operating manual is properly observed, up to 500 charging cycles may be carried out.

How many dispensing cycles can be carried out with a 25 ml volume?

This depends on the dispensing technique used. For liquid dispensing without a pump, it is possible to carry out 2,000 cycles. With a pump, only 650 are possible.

How is the wall holder fastened to the wall?

The wall holder has a Velcro® strip that is pressed onto the wall after the protective film has been removed. The wall holder can be pulled off sideways from the Velcro.

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Faq for biophotometere and uvettes

BioPhotometer:

Does room light effect the reading when using the BioPhotometer? Do you need to cover your sample?

No, the detector and the light source are recessed so room light does not affect the measurements taken by the BioPhotometer, so you do not need to cover your sample.

How can I improve the consistency of my results when I am using the BioPhotometer?

If you observe inconsistency in results, double check that your sample is thoroughly mixed. DNA or RNA solutions may be viscous. If the solution is not homogenous then OD values can vary from reading to reading. It is important to mix your sample in a separate tube and then transfer it to the cuvette. Especially with small sample sizes, mixing in the cuvette can be inefficient. In addition, spinning down your DNA or RNA solution prior to taking any measurements can eliminate particulates, which also could cause erroneous readings.

Why can’t I print from my BioPhotometer?

The dip switch settings on the printer are most likely set incorrectly. There are three dip switches and each switch contains 8 switches within. To confirm that the settings are correct call or e-mail Eppendorf for a copy of Seiko thermal printer switch settings. If your dip switch settings are correct, try changing the cable. Many times a faulty cable can interrupt communication between the two units.

What is the warranty period for the BioPhotometer?

The BioPhotometer has a warranty period of two years.

Which cable can be used to connect the BioPhotometer and the Thermal Printer?

The Thermal Printer is connected to the BioPhotometer using the Eppendorf Printer Cable (order no. 0013 610.517).

What are the dimensions and weight of the DPU-414 printer?

Dimensions of the DPU-414 printer are 160 mm x 70 mm x 170 mm (W x H x D). It weighs approximately 400 g.

What is the light-path height of the BioPhotometer?

The light-path height of the BioPhotometer is 8.5 mm.

How many calibration curves can be stored per protein-determination method (Bradford, Lowry, BCA)?

One calibration curve can be stored per method. In addition, it is possible to save a second curve under the “micro” method.

What is the smallest amount of DNA that can be reasonably measured?

The detection limit can be derived from the possible measuring error. An error of <= 0.003 Absorbance units can be expected, which is equivalent to a concentration of 0.15 µg/ml dsDNA. For example, if an absorbance value of approximately 0.050 is measured at 260 nm, then we know the concentration to be 2.5 µg/ml dsDNA, (because A is multiplied by the factor of 50 as the known standard and is preprogrammed for dsDNA). Therefore, the concentration determined from the measured value is between 2.35 and 2.65 µg/ml utilizing an error rate of 0.003 A (2.5 µg/ml ± 0.15 or 6%). Therefore, the amount of error that you will allow in your application will determine what the lowest amount of DNA you can reasonably measure. As the actual concentrations decrease, the error rate of 0.003 A becomes a higher percentage of the reading.

Can cuvettes from other manufacturers be used?

Yes, on condition that the specific dimensions of the cuvettes enable them to fit into the BioPhotometer and that they have a measuring window in line with the light-path height of the BioPhotometer (8.5 mm).

Is the OD 600 method a linear method? Which influences must be taken into account?

The measuring method is non-linear when the absorption curve is monitored across wide OD ranges and dilutions. The values are organism-dependent (size, shape) and device-dependent (light path geometry, proportion of scattered light rays measured). Therefore a calibration curve for each organism and for each device must be determined based upon the actual cell number. When the curve is known, it is possible to carry out measurements in those areas that run virtually linearly. In the course of one working day, the BioPhotometer is used repeatedly at irregular intervals.

Do you recommend that the device be switched on and off several times throughout the day or should the BioPhotometer be ready to measure at all times?

The operating life of the xenon lamp is affected neither by switching it on and off several times a day nor by leaving it on for several hours at a time. For this reason, users may decide for themselves whether the BioPhotometer should be switched on and off several times or should be left running permanently. The xenon lamp is “”active”” during measurement only; it then transfers to a Standby mode.

Is it possible to perform quantitative measurements using the BioPhotometer?

Yes. DNA/RNA/Oligos are quantified at 260 nm (UV), whereas proteins are measured either directly at 280 nm or with colorimetric assays, such as Bradford, Lowry or BCA. In addition, bacteria suspension can be determined at 595 nm (OD 600).

How do I set up the DPU 414 Printer for the BioPhotometer?

Download this PDF which contains parameters for DPU set up. ( PDF 72k)


UVettes

Can I reuse the UVettes?

Eppendorf recommends that the UVette be discarded following the sample measurement in order to guarantee contamination-free analysis. Cleaning processes and/or multiple use of an UVette may damage the quality of the optical surfaces, particularly in the sensitive UV-range. Even the slightest changes in the measuring area affect the quality of the results, even though this is not immediately visible.

When do you recommend using the 2 mm light path of the UVette versus the 10 mm?

In general, the absorbance should be within the linear measuring range of the BioPhotometer (OD: 0.0 –3.0). As a rule, users work with an absorbance of about 2.0. When working with a higher concentration sample that has an absorbance greater than 2.0, we recommend measuring using the 2 mm light path. For samples with a normal or a lower concentration, the 10 mm light path is suitable.

Should the same UVette be used for the measurement of the blank and of the sample?

The same UVette must be used for both measurements, since the self-absorption of every UVette is different. Using the same UVette guarantees that exact values are obtained.

Is a transmission curve (self-absorption of the UVette at different wavelengths) available for the UVette?

Yes, this can be found in the pack insert of the UVette. Copies are available from our Application Support ([email protected]).

What is the UVette made of?

The UVette is made of a UV-transparent plastic. It is free of fluorpolymers and other halogenated hydrocarbons, and is suitable for all commonly used methods in the fields of molecular biology and biochemistry.

Which reagents is the UVette resistant to and which reagents is it not resistant to?

Resistant to

Acids, for example:
  • acetic acid 96%
  • perchloric acid 10%
  • nitric acid 65%
  • hydrochloric acid 36%
  • sulfuric acid 40%
  • trichloroacetic acid 40%
Alkaline solutions, for example
  • ammonia solution 25%
  • sodium hydroxide solution 20%
Alcohol, for example
  • ethanol
  • isopropanol
  • methanol
And other important reagents, for example
  • acetone
  • DMSO 10%
  • formaldehyde 40%
  • water-saturated phenol
Non-resistant to non-polar solvents, for example
  • gasoline
  • chloroform
  • diethyl ether
  • hexane
  • oleic acid
  • petroleum ether
  • carbon tetrachloride
  • toluene
*All substances were tested at room temperature over a period of 24 hours.
What are the minimum and maximum filling volumes of the UVette?

The minimum filling volume of the UVette is 50 µl and the maximum is 2,000 µl. Make sure that there are no air bubbles in the solution, and that there is no residual liquid on the inner wall of the UVette. For this reason when pipetting the minimum volume, it is best to pipette the 50 µl directly into the lower part of the UVette.

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Electroporation F.A.Q

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!