Learning to use a micropipette correctly is one of the first hands-on skills taught in any wet lab, because even small errors in pipetting technique can throw off every dilution and concentration calculated afterward. A micropipette is designed for precise, repeatable transfer of small liquid volumes, typically from about 0.1 microliters up to 1000 microliters depending on the model.
Choosing the Right Pipette and Tip
Micropipettes are generally sold in fixed volume ranges, each with a different accurate working range. Using a pipette near the bottom of its range is less accurate than using one suited to the volume you actually need, so a 5 microliter transfer should use a smaller-range pipette rather than the low end of a much larger one. Always match the tip size to the pipette model, since tips are not universally interchangeable between pipette brands or ranges.
Setting the Volume
Most adjustable micropipettes display volume on a small dial or digital readout. Dial to the desired volume carefully and confirm the number before drawing up liquid, since setting a pipette past its maximum range can damage the internal mechanism and ruin its calibration.
The Two-Stop Plunger Technique
A micropipette plunger has two distinct stops. Pressing to the first stop and then releasing draws up liquid equal to the volume set on the dial. Pressing further, to the second stop, expels any remaining liquid from the tip after the first dispense. Learning to feel the difference between these two stops, rather than pressing straight through in one motion, is the single most important technique for accurate pipetting.
Drawing Up Liquid Correctly
Hold the pipette vertically when drawing up liquid, and submerge the tip only a few millimeters below the surface, since submerging it too deeply increases the amount of liquid clinging to the outside of the tip. Depress the plunger to the first stop before inserting the tip, then release the plunger slowly and smoothly to draw liquid up; releasing too quickly can introduce air bubbles that make the drawn volume inaccurate.
Dispensing Correctly
When dispensing, touch the tip gently against the inside wall of the receiving container and depress the plunger smoothly to the first stop, pause briefly, then continue to the second stop to expel the final residual volume. Pulling the tip away before fully reaching the second stop leaves liquid behind and causes under-delivery.
Keeping the Pipette Vertical
A micropipette should be held as close to vertical as practical while drawing up or dispensing liquid. Tilting the pipette at an angle changes the effective volume drawn, especially at small volumes, because the geometry of the air cushion inside the tip shifts slightly.
Once liquid volumes are measured accurately, checking the resulting concentration with a Dilution Calculator helps confirm that the planned dilution matches what was actually pipetted.
Pre-Wetting the Tip for Better Accuracy
For the most demanding work, many technicians draw up and expel the target liquid once before taking the actual measured aliquot, a step often called pre-wetting the tip. This equalizes the thin film of liquid that clings to the inside wall of a brand-new, dry tip, which can otherwise cause the very first draw from a fresh tip to be slightly less accurate than subsequent draws with the same tip.
Routine Care
Store pipettes vertically on a stand when not in use, never lay them down with liquid still in the tip, and have them calibrated periodically according to the manufacturer’s recommended schedule, since drift in calibration is often invisible until it shows up as inconsistent results.
Matching Technique to Viscous or Volatile Liquids
Water-based buffers behave predictably in a micropipette, but viscous liquids and volatile solvents need small adjustments to the basic technique. Viscous liquids should be drawn up and dispensed more slowly than water, with a brief pause at each stop to let the liquid fully settle in or clear the tip. Volatile liquids, which evaporate quickly, are often pre-wetted more than once and dispensed promptly after drawing, since vapor loss inside the tip can otherwise make the delivered volume slightly smaller than intended.
Reading the Meniscus When Verifying by Eye
For larger micropipette volumes dispensed into a graduated container, it can help to visually confirm the liquid level against the container’s markings, reading from the bottom of the curved liquid surface, known as the meniscus, at eye level rather than from above or below. This is a secondary check rather than a substitute for correct pipetting technique, but it can catch an obvious error, such as a clogged or poorly seated tip, before the liquid is used further.
Frequently Asked Questions
Why does my pipette sometimes under-deliver liquid?
Common causes include not fully depressing to the second stop when dispensing, using a damaged or poorly seated tip, or drawing liquid up too quickly and introducing air bubbles.
Can I use any tip with any pipette?
No, tips are designed to fit a specific pipette brand and volume range, and a poor seal between tip and pipette shaft will cause inaccurate and inconsistent volumes.
Why is pipette angle important?
Holding the pipette at an angle other than vertical changes how liquid is held in the tip and can alter the effective volume, particularly for very small volume transfers.
