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Laboratory Pipette F.A.Q and Troubleshooting

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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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