Introduction to Pipetting

Reagents

  • Pipetting Introduction Bag

Consumables

  • Transfer Pipette (1)
  • Pipette Tips (3)

Equipment

  • Micropipette
  • Beaker filled with water

Abstract

This guide is the second chapter in the Biotechnology 101 kit, that teaches you the basics of hands-on molecular biology. The pipette is an indispensable tool when working with DNA and analysing genes, so you will need to be able to pipette with skill and confidence, before starting the research projects in this kit.

There are many different kind of pipettes. This is an introduction to transfer pipettes and variable micropipettes.

Guide

  1. Types of pipettes

    You will be using two types of pipettes during the Biotechnology 101 kit. You have an adjustable micropipette (1), and a bag of disposable transfer pipettes (2).

    Do not try to dial the pipette beyond 20μL, as this might break the pipette or cause it to become unreliable in its volume.

  2. Disposable Transfer Pipettes

    Transfer pipettes are made from squishy plastic. They are not designed to measure specific volumes, so they are generally used for larger volumes where accuracy is not so important. ‘Large’ in the context of molecular biology are volumes between 0.2mL and 10mL. The transfer pipettes provided measure 1 – 5 mL.

    Transfer pipettes are simple to operate. In general during these guides, they will be used when you need to transfer a larger amount of liquid. In these cases, it is important to watch the volume, and to suck up the liquid slowly.

    If you have never used this type of pipette before, give it a try and pipette some water between different cups. Get a feel for different volumes and try to pipette as precisely as you can, e.g. 1mL or 0.5mL.

  3. The Micropipette: An Overview

    Micropipettes are probably the most used tool in the laboratory.

    They are used to transfer precise, very small volumes of liquid. Their units are generally measured in microlitres (μL) which is one thousandths of 1 mL.
    The micropipette in the Biotechnology 101 Kit is a 2-20μL adjustable micropipette, so you can set its volume between 2 and 20μL.

    Adjustable micropipettes are more complex than a simple disposable pipette.

    Read this guide carefully to familiarise yourself with the correct operation of the pipette. This skill will be crucial to your success with lab experiments. There are also some important things to follow to ensure that you do not damage the pipette.
    There are several parts of interest: The micropipette has a pipetting button (1), and an eject-plunger (2). There is a dial that shows the currently set volume (3). Finally, there is the front of the pipette, onto which the pipette tip (4) attaches.
    In fact, you do not actually pipette liquids directly into the pipette. This would damage the pipette. Instead, every time you pipette, you use a new sterile pipette tip, and the liquid will be held by the pipette in the tip.

    Never suck up liquid directly into the pipette!
    Always use a tip, and make sure that the liquid never gets sucked up beyond the tip into the pipette itself.

    You have one box of pipette tips (5) in the Biotechnology 101 Kit, which is enough for all the projects in the kit.

    One box of pipette tips might look like a lot, but consider this: A pipette tip can only be used once. After you have used the pipette tip, it is contaminated by liquid you are pipetting. Usually you dispose of that tip and use a new pipette tip for the next sample. You will learn more about this throughout the kit.

    Make sure to close the box of pipette tips after you have taken a tip, to decrease the chance of contaminating the tips with accidental spills, etc.

  4. Adjusting the pipette

    You can change the volume that the pipette transfers by rotating the pipetting button.
    However, avoid dialling the pipette beyond its limit of 20μL.

    The dial increases in 0.5μL steps.

    Do not try to dial the pipette beyond 20μL, as this might break the pipette or cause it to become unreliable in its volume.

  5. Handling a micropipette

    Operating an adjustable micropipette involves a few more steps compared to a simple transfer pipette.

    The pipetting button on the adjustable pipette has two stops. Take the pipette and push the button slowly to feel the two different stops.

    The position of the first stop is directly related to the volume setting.

    Set the pipette to 5μL and feel the position of the stops. Now set the pipette to 20μL and note the difference. At 20μL, the first stop is at the lowest position possible, since it is the maximum volume it can dispense.

    What should I do if I have been winding the pipette past 20μL?

    Don’t worry if you accidentally overwind the pipette a couple of times. There is a risk of uncalibrating the pipette if you overwind it frequently past 20μL. In this scenario, you may notice a change in pressure when you press down, and you will need to replace the pipette.

    The function of the first stop is to draw up the liquid into the pipette tip in the set volume.

    To do this, you press the pipetting button down onto the first stop. Then you insert the pipette tip into the liquid you want to draw up, and release the pipetting button.

    Pressing down to the second stop ensures that all the liquid is dispensed.

  6. Practice Time

    Time to get familiar with the micropipette.

    To prepare, fill the glass beaker with tap water.

    Set your pipette to 15 μL

    It is good etiquette to set the pipette to the correct volume before attaching a pipette tip. This is in order to reduce the risk of contaming the tip once it is attached to the pipette.

    Open the box of pipette tips, and put on a pipette tip.

    Press the plunger down to the first stop, then put the tip into the water. When the tip is inside the water, release the button to draw up the water.

    How far should I insert my pipette tip when drawing up the sample?

    When beginning to pipette, you should aim to take the sample from roughly halfway into the sample. Generally, pipetting from the bottom of the sample will reduce the accuracy of the volume drawn up, and you also may block the tip if you press the tip against the bottom. As you become more experienced, try pipetting closer to the surface of the sample. As a rule of thumb, in terms of accuracy, the optimal depth to draw the sample from is just below the surface.

    This is the last chance to check if you have set the pipette to the correct volume. It is always a good idea to double check at this stage if the pipette is set to the intended volume.

    Now dispense the liquid again into the beaker, by pressing the pipetting button all the way down to the second stop. Do not release the pipetting button until you have taken the pipette tip out any liquid.

    Never release the pipetting button from the second stop when the tip is immersed in liquid. This would draw up liquid beyond the maximum volume, which could be more volume than the tip can fit. In that case, the liquid could reach into the pipette itself, which can end up damaging it.

  7. 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 touch a sample with the tip, because DNA from the sample could now be on the tip. If you keep using the same tip, you might contaminate other samples, as well as your reagents.

    In many laboratory workflows, you will need to change tips so often, that a micropipette has a specific ejector button for this. It will shoot the tip off of your pipette. In the lab, this happens a lot.

    Try it yourself now and shoot off the pipette into a container, such as a cup. Never aim your pipette tip at a person when ejecting it.

  8. More Pipetting Challenges

    It can not be overstated how important good pipetting skills are for successful lab work. Your experimental results in this kit will in part depend on your skill with the pipette.

    To finish, here is a little pipetting challenge designed to build your muscle memory. For this, keep using the same tip you have been using so far. If you just ejected it, take it back and put it back on the pipette. In the later experiments, you will need to change tips frequently to avoid contamination, so it’s best not to waste any now.

    For this challenge, you will need the petri dish and the tubes with dyes from the ‘Pipette’ bag.

    The Bento Lab tube rack might also come in handy for this activity. You can use the rack to hold the tubes containing the dyes.

    Open up your petri dish. You will use the petri dish as a surface to pipette onto. From the different colours, pipette different droplets onto the petri dish. For example, try to pipette uniform sized droplets. You can also experiment to see how it feels to pipette different volumes, and how they look on the petri dish. How large does the difference between 1 and 20μL feel?

    There is no right or wrong way to complete this practice challenge. Important is, that you get a feel for the micropipette, and train your muscle memory. This is a good opportunity to get creative and experiment with the colours, volumes and patterns.

    Try out a few pipette tips – you can use the beaker as a bin. But don’t use too many! You will need them later. Using up to 5 pipette tips for this activity is a good idea.

    How often should I change the pipette tip?

    You only need to change the tip if the tip becomes contaminated by touching anything that is not the sample you are trying to pipette.

    Some of my sample is left in the tip even after I eject down to the second stop. What should I do?

    Don’t worry if there is only a thin ‘ring’ of the sample, or tiny droplets remaining in the tip. If lots of sample remains, then we would recommend you repipette the sample back up, and then start again. If you are unsure, or if the liquid volume needs to be accurate, the safest option is to start again.

    How far from the surface should I pipette out the sample?

    Generally, it is best to pipette as close to the surface as possible, i.e. ~2mm away. You don’t want the pipette tip to touch the surface, or to pipette out so far away, you lose control. Try out pipetting from different heights and pay attention to what distance gives you the best control.

    When I dispense the sample, sometimes air bubbles form on the surface. Is this normal?

    Air bubbles sometimes form when you are ejecting the sample down to the second stop, especially if the pipette tip breaches the part ejected sample. Don’t worry about air bubbles in this activity or when pipetting samples and reagents into each other as the air bubbles will quickly pop. Bubbling is only a problem if you are planning to reuse the tip for pipetting the same reagent again or if it is crucial that the sample does not dissipate to other areas of the container (e.g. pipetting into wells during gel electrophoresis).