Pipetting Protocol

Introduction

The pipette is an essential tool for nearly anyone in molecular biology. You’ll use a pipette in many scenarios to accurately dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working in a laboratory, properly dispensing liquid ensures the accuracy of your experiment and any changes to the amount you are dispensing can negatively impact your experimental results. This protocol will guide you in selecting the correct pipette and pipette tip and using the pipette.

Last Update: September 2022

Equipment

• Pipettes
• Pipette tips
• Waste container
• Containers to hold measured liquid (ex: microfuge tube, bottle, etc.)
• Labels for containers

Reagents

• Liquid for pipetting

Background Information

Anatomy of a Pipette

This front view photo of a P1000 pipette shows the plunger at the top, which is used to draw and expel liquid into the pipette tip, the volume display which shows the set volume for the the pipette, and the tip ejector ring at the bottom which pushes the pipette tip off of the pipette.

This side view photo of a P1000 pipette shows the volume adjustment ring right below the plunger. This ring changes the pipette volume. Below that is the tip ejector button which is used to push the tip ejector down the pipette to remove the tip.

Choosing a Pipette and Pipette Tips

Although there are many different types of pipettes that have different mechanisms to disperse liquids, this protocol is focused on single channel pipettes. These come in standard sizes: P2, P10, P20, P200, and P1000. The table shows the pipette with the appropriate dispense volume:

This image shows the P2, P10, P20, P100, P200, and P1000 as shown from left to right.

Pipette	Dispense Volume
P2	0.2 to 2 µL
P10	1 to 10 µL
P20	2 to 20 µL
P100	10 to 100 µL
P200	20 to 200 µL
P1000	100 to 1,000 µL

Each pipette should be used with the appropriate tips, which are made in a number of sizes and colors depending on the pipette they are used with and the volume to be dispensed. The boxes that the tips come in often indicate a volume range that the tip can hold. This should give you an idea of what pipette to use with it.

The purpose of the pipette tip is so that the same pipette can be used for measuring different samples without cross contamination as long as the tip is changed between samples. Tips can come loose in a bag, or can come preloaded into tip boxes. In either case, once assembled into their boxes, pipette tips can be sterilized in an autoclave as needed to prevent contamination. When not in use, the tip box should be closed to prevent contamination.

Reading the Volume on the Pipette

Although each pipette performs the same function, the numbers are read differently on each type of pipette. This section will help you understand how to read each display to dispense the correct amount of liquid reagent.

P1000: The first red digit is thousands of µL, the middle is hundreds, and the third is tens. Therefore, 1000 µL would read as 100 from top to bottom (as shown in the picture above), while 650 µL would read as 065.

P200: The first digit is hundreds of µL, the second is tens, and the third is ones. Therefore 100 µL would read as 100 from top to bottom (as shown in the picture above), while 95 µL would read as 095.

P20: The first digit is tens of µL, the second is ones, and the third (red) digit is tenths. Therefore, 10 µL would read as 100 (as shown in the picture above), while 2.2 µL would read as 022.

P2: The first digit is ones of µL, the second red digit is tenths, and the third (red) digit is hundredths. Therefore, 1 µL would read as 100 (as shown in the picture above), while 0.5 µL would read as 050.

Each volume display on these pipettes will also have small tick marks at the bottom. For the P1000, this represents the ones. For the P200, this represents the first decimal place. For the P20, this represents the second decimal place, and for the P2 this represents the third decimal place.

Procedure

How to Pipette

Now that you know the different parts of the pipette, how to select tips, and how to read a pipette, you’re ready to begin using the pipette. To the right is a photo of how you might hold a pipette.

1. Without moving past the maximum possible setting on the pipette, set the desired volume of the liquid on the pipette. Depending on the pipette you’re using, you will be rotating dials behind the numerical display or the top of the plunger of the pipette.
2. Select the pipette tip that is designed to fit your pipette. To load a tip onto a pipette, open the tip box and push the end of the pipette onto the tip. Do not touch the tips with your fingers to avoid contaminating the tip or puncturing your glove. Ensure that the tip is properly set on the pipette to ensure pipetting accuracy.
3. Once the pipette is set and you have the liquid you wish to pipette ready, push down the plunger. There will be multiple “stops” on the plunger, where you feel resistance while pressing down on the pipette. Stop at the first “stop.” The first stop expels enough air from the pipette for an accurate measurement in the next step. The second stop is meant to expel excess liquid from the tip once you’ve reached step 8.
4. Continuing to hold down the plunger, gently lower the pipette tip into the liquid. Do not submerge the pipette itself into the liquid and avoid touching the pipette to the inside of the tube. Only the tip should be in the liquid.
   Pro-Tip
   If pipetting from a relatively large container holding a small volume of liquid, tilt the container holding the liquid so that it's easier for the pipette tip to reach the liquid. Aim to let only the pipette tip into the container in order to avoid contaminating the contents.
5. Once the end of the tip is submerged, slowly release the plunger, allowing it to go back to its resting position.
6. Wait for the liquid to stop flowing into the pipette tip. Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower the flow rate into the pipette tip.
7. Once the liquid has finished drawing into the pipette tip, lift the pipette so that the tip exits the liquid. When you lift the pipette tip out of the liquid, no liquid should drip from the tip. This could indicate that the tip is not on the pipette properly.
8. Place the pipette tip into the container you are using to hold the measured liquid (ex: another microcentrifuge tube, a bottle etc.). If there is already other liquid in the receiving tube, place the pipette tip into the liquid. If this is a new container, place the tip near the bottom of and gently in contact with the container (capillary action between the container and the liquid will help draw the liquid out of the pipette tip). Then, slowly press down on the plunger all the way to the second stop to release the liquid.
   Pro-Tip
   Dispense until the first stop, wait, and then dispense to the second stop to expel any residual liquid in the tip.
9. Before releasing the plunger, lift the pipette tip out of the liquid. Once you are clear of the liquid you can slowly release the plunger. Remove the entire pipette from the container making sure not to touch the sides of the container with the pipette.
10. Discard the pipette tip by holding the pipette tip over your dedicated waste container and pressing on the tip ejector button. If pipetting biohazardous materials, be sure to discard into a biohazardous waste container.

Conclusion

Pipetting accurately and precisely is important since even small changes in the volume could affect your experiments. Beyond knowing the proper way to pipette, it’s important for scientists to calibrate their pipettes to make sure that they are properly tuned to dispense the correct amounts of liquid.

If you are pipetting the same amount of liquid into different tubes or into wells of a microplate, you may consider using a multichannel pipette. Please watch Addgene’s protocol on multichannel pipetting to learn more.