Pipetting 101

Activity 1
How to operate and handle a micropipette

Abstract

In this activity you will learn the how to operate a micropipette, including setting volumes and attaching tips.

Materials

Included in Biotech 101 Kit

1 20 µL micropipette

2 20 µL pipette tips

3 beaker filled with tap water

Additional Equipment

4 container for ejected tips

Guide

  1. Introduction

    Micropipettes are used to transfer very small and precise volumes of liquid.  Their units are generally measured in microlitres (µL) which is one thousandths of 1 mL. 

    1 µL is the same volume as 1 mm3, and 1 mL or 1000 µL is the same as 1 cm3.

    This kit uses a 20 µL adjustable micropipette.
    Other commonly used pipette volumes are 10 µL, 100 µL, 200 µL, 1000 µL, or even 5 mL.

    The micropipette works by air displacement: a small vacuum is created when the pipetting button is pressed down and then released. This draws up a very small volume of liquid into the disposable pipette tip. The liquid can then be expelled by pressing down on the pipetting button.

    You should never pipette liquids directly into the pipette shaft. Instead, every time you pipette, use a new sterile pipette tip.
    The liquid will be held by the pipette in the tip.

    In this activity you will learn how to adjust the pipette to a specific volume, attach pipette tips, and pipette water.

  2. Setup

    First, fill your beaker (or a cup) with tap water. You will also need your pipette and a box of pipette tips.

  3. Adjusting the pipetting volume

    Holding the pipette as in the photo below, rotate the pipetting button at the back of the pipette until the volume dial in the body shows 15 µL. 

    Do not to turn the volume lower than 2 µL or higher than 20 µL.
    Forcing the pipetting volume beyond its design limits may damage the pipette, or even break it.

    Note that the graduations in the dial are restricted to 0.5 µL intervals, so any volumes will have to be rounded up or down to the nearest 0.5 µL. For more precision a smaller volume pipette would be needed.

  4. Attaching pipette tips

    To attach a pipette tip, first hold the pipette vertically above a pipette tip.
    Then press down gently until the shaft is inside the pipette tip.

    Move the pipette up and down gently twice in a tapping motion to seat the tip securely. Try not to push down too hard or for too long.

    To help guide the shaft into the tip, you may find it useful to stabilise the shaft against a finger of your other hand while moving it.

  5. The First and Second Stops

    The pipetting button at the back of the adjustable pipette has two stops. These stops are important for accurate and precise pipetting. 

    Hold the pipette and push the button slowly to feel the two different stops. You will feel your thumb come to a stop (the first stop), but if you apply more pressure you can push the button down until it can go no further (the second stop).

    The distance from the released pipetting button to the first stop determines the volume pipetted up or dispensed. The distance from the first stop to the second stop is for blowing out all the liquid in the tip.

  6. Aspirating liquids

    To pipette liquids into the pipette tip:

    1. Press the pipetting button to the first stop and hold it there
    1. Insert the tip a few mm under the surface of the liquid
    1. Release the button slowly to draw the liquid into the tip
    1. Remove the pipette tip from the liquid

    You have now aspirated (i.e. pipetted up) 15 µL into your pipette tip.

  7. Expelling liquids

    After you have aspirated liquid into the pipette tip, it is time to expel the liquid again. 

    1. Place the end of the pipette tip just a few mm under the surface of the beaker of water.
    1. Press the pipetting button slowly to the first stop to expel most of the liquid.
    1. Press the pipetting button slowly to the second stop to expel any remainder.
    1. Move the pipette tip out of the water and briefly touch the pipette tip to the beaker wall to remove any droplets still attached to the tip.
    1. Slowly release the pipetting button.

    You will now have expelled 15 µL of water back into the beaker. Repeat the aspirating and expelling process several times more until you can do it smoothly and easily.

  8. Steadying the pipette while pipetting

    You may have noticed that when you hold a pipette with one hand the tip can sometimes be a little shaky and difficult to control.

    This can be a problem while pipetting in molecular biology, because you will need very fine control to  needed to transfer very small volumes of liquid from one small tube to another, or into the narrow wells of an electrophoresis gel.

    An easy solution is to treat a pipette as a two handed tool, with one hand holding the handle, and the other hand  acting to stabilise the shaft (not the tip), like you would support a snooker or pool cue.

    By providing this extra bit of support you can easily reduce wobbles and mispipetting, and pipette with greater accuracy, confidence and speed. 

  9. Ejecting the Tip

    In an actual experiment, after you have used the tip, you usually need to dispose of it. You do not want to use the same tip again after you have touched reagents or a sample with the tip because of the risks of cross contamination. This can be a real risk if using expensive reagents or valuable samples.

    The need to change tips frequently has led to a dedicated ejector button being included at the back of most micropipettes. This will eject the used tip with some force.

    Try it yourself now and eject the pipette into a container, such as a plastic cup or pot. 

    But never aim your pipette tip at a person when ejecting it!

  10. Reusing Pipette Tips

    For real scientific work you have to use a fresh tip every time to  avoid contamination of your sample and reagents , and to restrict the spread of any potentially harmful substances r biological materials if these are being used..

    However, for practice pipetting we recommend keeping a box of pipette tips aside that you label clearly, and wash and reuse the tips. 

    If you are reusing tips for practice, it is still a good idea to pretend you are using a new tip each time to establish good muscle memory when pipetting.
    To practice, you can do most of these activities with a single or a small number of tips, rinsing them between different dyes by pipetting up and down in a beaker or plastic cup of water. You can also develop your muscle memory for using new tips by rinsing a tip after use, placing back in the box, and then reattaching it as if it was a new tip.

    Single-use plastics are a major global issue, and there is active development of reuse and recycling of laboratory plastics in many institutions. If in the future you find you are producing very large amounts of lab plastics waste we recommend that you investigate whether there are any such initiatives in your area that you could participate in.