Biotech Explained · Life Sciences Careers
How to Use a Micropipette: Steps, Checks and Common Mistakes
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A micropipette moves liquid in microliters, the thousandths of a milliliter that molecular biology, cell work and diagnostics run on. It looks simple: a plunger, a dial and a disposable tip. Yet most volume errors in a teaching lab, and plenty in research labs, come from how the plunger is pressed and released rather than from the instrument itself.
This guide covers the parts, the standard technique (forward pipetting), the variant for sticky liquids (reverse pipetting), how to choose pipette tips, when a serological pipette is the better tool, and a ten-minute check with water and a balance that tells you whether your hands are accurate.
The parts you will touch
Most adjustable micropipettes are air-displacement instruments. A piston inside the shaft moves a cushion of air, and that air pulls liquid into the disposable tip. The liquid never touches the pipette itself, which is why the tip is the only part you change between samples.
- Plunger button: the top button you press with your thumb. It has two stops.
- Volume adjustment: a knob, or the plunger itself, turns to set the volume. Many models have a lock.
- Volume display: a small window with three or four digits.
- Tip ejector: a separate button that pushes the used tip off.
- Shaft and tip cone: the barrel and the tapered end the tip fits onto.
The two stops matter more than anything else on that list. Press the plunger gently and you meet resistance: that is the first stop, and the travel to it corresponds to the volume you set. Push past the resistance and you reach the second stop, the blow-out, which pushes out the last film of liquid. You aspirate (draw up) from the first stop and dispense all the way to the second.
Pick the right size for the volume
Accuracy is best in the upper part of a pipette's range and worst near the bottom, so use the smallest pipette that covers your volume. Labs usually call them by their top volume: P20, P200, P1000.
| Common size | Typical range | Good for |
|---|---|---|
| 2 µL | 0.2 to 2 µL | enzymes, primers, tiny reagent volumes |
| 10 or 20 µL | 0.5 or 2 to 20 µL | PCR set-up, loading gels |
| 200 µL | 20 to 200 µL | buffers into tubes, plate wells |
| 1000 µL | 100 to 1000 µL | larger transfers, resuspending pellets |
| 5 or 10 mL | 0.5 or 1 to 10 mL | bulk reagent additions |
The display reads differently on each size. On many 20 µL models the digits 1, 2, 5 mean 12.5 µL, while on a 200 µL model the same digits mean 125 µL. Check the markings or the manual for your model before trusting a reading, and never turn the dial past the top or bottom of the range, which can damage the mechanism.
What you need
- An adjustable micropipette sized for your volume
- Tips that fit it, sterile filter tips if the work is PCR, cell culture or anything you must not carry over
- The liquid to transfer and a receiving tube or plate
- A pipette stand, a waste container for used tips, gloves
- For the accuracy check: distilled water, a small weighing vessel and an analytical balance reading to 0.1 mg
How to use a micropipette, step by step
This is forward pipetting, the default method for water-like solutions such as buffers, diluted reagents and most media.
- Set the volume. Turn the adjustment to your volume and lock it if your model has a lock. Some manuals ask you to overshoot slightly and come back when increasing the volume, so the mechanism settles; follow yours.
- Fit a tip. Press the tip cone firmly into a tip in the rack with a slight twist. Don't hammer it on. Banging the pipette onto tips wears the cone and can seat the tip at an angle, and a poor seal leaks.
- Pre-wet the tip. For the first draw of a new liquid, aspirate and dispense it two or three times back into the source. This brings the air cushion to the same humidity and temperature as the liquid and reduces the shortfall on the first dispense.
- Press to the first stop and hold it there. Do this before the tip enters the liquid. Pressing while submerged blows bubbles into the sample.
- Immerse the tip just below the surface, with the pipette close to vertical. A few millimeters is enough for small volumes; large tips go a little deeper. Going deep coats the outside of the tip with liquid that then rides along into your tube.
- Release slowly and smoothly. Let the plunger rise under your thumb rather than snapping back. Wait about a second for the liquid to finish rising, longer for big volumes or thick liquids.
- Withdraw and look. Pull the tip straight out and glance at it. An air bubble, or a droplet clinging to the outside, means empty it back and draw again.
- Dispense against the wall. Touch the tip to the inside wall of the receiving tube at an angle and press steadily to the first stop. Pause, then press on to the second stop to blow out the rest.
- Slide the tip up the wall and out while keeping the plunger down. Releasing it while the tip is still in the liquid sucks some back up.
- Eject the tip into waste, then put the pipette back in its stand, upright.
Reverse pipetting for viscous or foaming liquids
Glycerol stocks, concentrated protein, detergent solutions and serum stick to the tip wall or foam. For these, press straight to the second stop before you immerse, release to draw up a slight excess, then dispense only to the first stop. The excess stays in the tip and goes to waste with it. Reverse pipetting is also handy when you dispense the same volume into many wells from one draw.
Pipette tips: which kind
Tips are not interchangeable details. The wrong one is a common source of drips and carry-over.
- Standard tips suit most routine work, sold loose in bags or in racks. Racked tips are easier to keep clean and to fit squarely.
- Filter (aerosol barrier) tips have a plug that stops aerosols and splashes reaching the shaft. Use them for PCR, cell culture, and infectious or radioactive material.
- Low-retention tips have a treated inner surface, so less liquid stays behind with detergents and proteins.
- Gel-loading and extended-length tips reach into narrow wells and deep tubes.
Sterile tips come pre-racked and sealed. Color conventions (yellow for 200 µL, blue for 1000 µL) are common but not universal, so read the box.
When a serological pipette is the better tool
A serological pipette is a long graduated tube, usually sterile, individually wrapped plastic, used with a motorized or bulb pipette controller. Common sizes run from 1 to 50 mL. Use it whenever you are moving whole milliliters: medium into flasks, buffer into a conical tube, resuspending a pellet in 10 mL. A 1000 µL micropipette can move 5 mL in five draws, but every draw adds error and every extra tip adds a chance of contamination.
Read a serological pipette at eye level, at the bottom of the meniscus. Check the markings near the top too: many are designed so the last drop is blown out and some are not, so the same graduation can deliver slightly different volumes.
Common mistakes and what they look like
| What you notice | Likely cause | Fix |
|---|---|---|
| Volumes run low, especially the first | tip not pre-wetted, plunger released too fast | pre-wet, release slowly |
| Bubbles in the tip | tip too shallow, or plunger pressed while submerged | press to the first stop before immersing |
| Drops on the outside of the tip | tip went too deep | immerse only a few millimeters |
| Liquid creeping back after dispensing | plunger released before the tip left the liquid | keep it pressed until the tip is out |
| Dripping tip | poor seal, wrong tip, volatile liquid | reseat or change tips, use reverse pipetting |
| Liquid inside the shaft | pipette laid flat with a full tip | store upright, clean as the manual says |
| Replicates disagree | changing angle or rhythm between draws | same angle, same pace every time |
One more that is easy to miss: holding a micropipette in your fist for an hour warms the air inside it, and warm air expands, so volumes drift during long runs. Put it in the stand between samples.
Check: a gravimetric test with water
You can check your own technique, and catch a pipette that needs service, with the weighing method that calibration labs build on (the international standard ISO 8655). Water has a density close to 1 g/mL, so 100 µL of water weighs close to 100 mg.
Set a 200 µL pipette to 100 µL. Put a small vessel with a little water in it on the balance (the water layer and a lid cut evaporation). Tare, dispense, record the reading, tare again, and repeat ten times. Note the water temperature.
Here is a worked set of ten readings, in mg: 99.4, 99.1, 99.6, 98.8, 99.3, 99.5, 99.0, 99.2, 99.7, 99.4.
- Mean mass: 993.0 / 10 = 99.30 mg.
- Convert to volume. ISO 8655 uses a correction factor, Z, for water density and air buoyancy. At about 20 °C and normal air pressure, Z is about 1.0029 µL per mg, so the mean volume is 99.30 × 1.0029 = 99.59 µL.
- Accuracy, or systematic error: (99.59 minus 100) / 100 = about minus 0.4 %.
- Precision: the standard deviation of the ten readings is about 0.28 mg. Divided by the mean, that is a coefficient of variation of about 0.28 %.
Compare both numbers with the specification sheet for your model at that volume. If your precision is poor but the pipette passes when a colleague runs the same test, the issue is technique. If you both get the same offset, the pipette probably needs calibration or service. Manufacturers publish a recommended calibration interval; most labs also run a quick check like this between calibrations.
Errors compound in a dilution series, which is why the serial dilution calculator shows the transfer volume for every tube: choose a pipette where each of those volumes sits in the upper part of its range. For a single dilution, the dilution calculator does the C1V1 arithmetic.
Bench essentials
- Adjustable micropipette, 100 to 1000 µLsingle channel · 100 to 1000 µL · autoclavableA single-channel workhorse for the volumes most buffer and dilution work uses; pair it with matching tips.Open on Amazon
- 1000 µL universal pipette tips1000 µL · universal fit · 500 piecesTips for the 100 to 1000 µL range that fit most common pipette brands.Open on Amazon
- 1.5 mL microcentrifuge tubes1.5 mL · 500 tubes · EO sterilizedSnap-cap tubes for aliquots, small dilutions and spins.Open on Amazon
Picked from specifications, manuals and published reviews; we have not tested these items ourselves.
FAQ
What is the difference between the first and second stop?
The first stop marks the calibrated volume and is where you start aspirating. The second stop is the blow-out, used when dispensing in forward pipetting to push out the last drop. In reverse pipetting the roles swap.
Can I use any brand of tip?
Universal tips fit many models, but fit varies. A good tip slides on with light pressure, sits straight and does not drip when you hold a full tip still for a few seconds.
How do I clean a micropipette?
Wipe the outside with 70 % ethanol or a cleaner the manufacturer approves. If liquid got into the shaft, follow the manual for taking the lower part apart. Some models allow autoclaving of the lower part or the whole pipette; check before you try.
Where are micropipettes used outside research labs?
Clinical and diagnostic labs, food and water testing, forensics and cell therapy manufacturing all depend on them. Our piece on a day in a cell therapy manufacturing lab shows the same pipetting habits under production rules.
This explainer is general information, not medical advice. Decisions about treatment, donation or trials belong with a clinician who knows your history.
