Several years ago as a freshman in a research lab, the very first project I received was to pipette incremental micro-volumes of H2O onto a piece of parafilm. Boring! Weighing the liquid on parafilm and comparing the weight between 10 replicates for each micro-volume continued for a week before I touched anything else in that lab! Fortunately, lack of funding helped me get out of that lab and into more interesting projects over the years. However, the importance of having a good technique for pipetting did not go without deliberation as I trained.
The pipette is that one instrument in the lab that can give different results based on the person using it. This is especially true when running quantification experiments.
Releasing the plunger too rapidly, not pre-wetting a new tip and not immersing the tip far enough are very common when working too quickly in the lab. Such mistakes can lead to differences in volume between samples, resulting in skewed assay results.
Here are 10 tips (from ARTEL) that may come in handy when training new students in your lab.
- Use the correct pipette tip – This is easy to tell! Use the right tips with the right pipette. Alternate brands are acceptable if they have been tested by the vendor to work with your pipette brand.
- Pre-wet the pipette tip – Pre-wetting increases the humidity within the tip, thus reducing evaporation.
- Examine the tip before dispensing the sample – When you immerse the tip too far into the liquid, there are often extra droplets sticking to the outside of the tip (for example, when pipetting enzymes). Make sure you wipe off the outside gently with a Kimwipe before dispensing. Take care not to touch the tip opening to avoid wicking away some of the required volume!
- Use standard mode pipetting – Remember to use the second stop on the pipette to dispense the entire volume.
- Pause consistently after aspiration – After drawing up liquid, make it a habit to pause before pulling out the tip completely. The liquid is still flowing into the tip even after the plunger stops.
- Pull the pipette straight out – Touching the sides of the container from which liquid is drawn will cause wicking and loss of volume.
- Use consistent plunger pressure and speed – “It is all about rhythm – repeatable actions produce repeatable results”!
- Immerse the tip to the proper depth – Large volume pipettes (1-5mL) should be immersed 5-6 mm below the meniscus. Smaller volume pipettes should be immersed 2-3 mm below the meniscus. Too much immersion will cause liquid to stick to the outside of the tip, and too little immersion will result in air bubbles.
- Minimize handling of the pipette and tip – The heat transferred from your hands to the pipette and/or the tips can affect delivery volumes.
- Work at temperature equilibrium – Working at a constant temperature minimizes variations in pipetted volumes. This is a bit tricky since many reagents require storage on ice, while the process of pipetting itself is done at room temperature! If your procedure does not allow temperature equilibrium, then there are more important tips that you should be taking care of, like #1, #3, #6 and #8.
More details on these ten tips can be viewed here.
If you are a visual learner, here is video for you to watch.