Pipetting
There are two main but related methods to pipetting, micropipetting for volumes of 1 ml or less and regular pipetting for volumes greater than 1 ml.
regular pipetting
For volumes 1 ml or greater, especially when the liquid in the container cannot be reached with just the sterile micropipette tip. Usually pipettes come in sizes of 2 ml, 5 ml, 10 ml, and 25 ml.
There are "thumb wheel" and "bulb" manual pipetters and electric pipetters. Currently we have green thumb wheel pipetters in the lab.
I used an electric chargeable Drummond Pipet-Aid (similar to this https://www.pipettes.com/drummond-scientific-pipet-aid-supplied-complete-with-110-v-recharger-ul-csa-approved-plus-four-replacement-filters-and-holster-wall-bracket) at the recent HHMI workshop and was impressed. It fit a range of transfer pipettes sizes and gave a range of control without being too heavy. Floyd (talk) 03:16, 17 July 2018 (PDT)
micropipetting
Most pipettes have a main button with a first stop and second stop and a second eject button. In general (if not "reverse pipetting" viscous liquid) transfers will be
- 1. Check volume
- 2. Add tip
- 3. First stop
- 4. Insert into container
- 5. Release
- 6. Transfer to new container
- 7. First stop-Second stop
- 8. Remove
- 9. Release
- 10. Eject tip
Here is each step with notes.
- 1. Check volume (Be aware of how different pipettes represent the decimal place. On some models this is a line and the volume is read top to bottom. Adjust the volume by smoothly rotating the dial. Do not "rake" the dial quickly; this can throw off the calibration.)
- 2. Add tip (Disposable pipette tips will usually be in a tip box. Lightly insert the end of the pipette and lift the tip out. If you accidentally touch the tip to a surface throw it away and get a new tip. The tip box lid should be kept closed between getting new tips.)
- 3. First stop (Pressing the plunger down there will be a point of light resistance. This prepares the pipette to draw the correct volume of liquid. Be careful to not press the plunger past the first stop at this stage.)
- 4. Insert into container (You should hold the container of liquid in your other hand so you can see the pipette tip and the liquid surface. Hold the pipette vertically. Insert the tip of the pipette into the liquid as little as possible.)
- 5. Release (Smoothly release the plunger to draw the liquid. Be careful about air bubbles, especially in small containers, and that you are not pipetting air. Visually confirm the liquid is being drawn continuously into the pipette tip. Don't "pop" the button up and release too quickly and abruptly. This can cause liquid droplets to fly from the tip into the pipette and contaminate later liquids.)
- 6. Transfer to new container (Be careful to not accidentally touch the pipette tip against any surface except the inside of the new container. Hold the new container in your other hand and touch the pipette tip lightly against the inside wall, holding the pipette at an angle (not vertically against the bottom which could inadvertently seal the tip).)
- 7. First stop-Second stop (Smoothly press the plunger down beyond the first stop (light resistance) to the second stop (firm resistance) and visually confirm that the liquid is going into the new container.)
- 8. Remove (Life the tip out of the container before releasing pressure; don't accidentally pipette the liquid back into the tip.)
- 9. Release (Smoothly release pressure so that tiny left-over droplets don't aerosol and contaminate the pipette.)
- 10. Eject tip (Hold the pipette over a waste tip collection container and press the tip eject button. Avoid "shooting" the tip at the container (this happens naturally as you get more practice and become tired); this can result in contamination.)
General points: Pipettes are not toys. Do not play with them shooting pipette tips at each other (for some reason students have an urge to do this when they first encounter a pipette). Avoid dropping the pipette or leaving them out. Pipettes should be regularly calibrated. Filter tips can be used to minimize contamination; however, these are more expensive and with proper technique they are not necessary except possibly in situations where extremely small levels of contamination can be critical (such as PCR). Balances are more accurate than pipettes. If ultra-precise volumes are needed use a pipette with a container tared on a balance to measure out the liquid (and be aware that it will change slowly with evaporation).
Size Ranges
Volumes in the 1 µl – 20 µl range should be transferred with a P20 micropipette.
Volumes in the 20 µl – 200 µl range should be transferred with a P200 micropipette.
Volumes in the 200 µl – 1000 µl range should be transferred with a P1000 micropipette.
In general micropipettes work best in the middle of their range (half of their "number", e.g., 100 µl for a p200). Do not pipette above the maximum range, liquid drawn through the tip and into or touching the end of the pipette results in contamination.
Volumes less than 1 µl can be tricky to work with. Use a P10 micropipette instead of a P20 and/or extended pipette tips if you have them.
Links
https://bitesizebio.com/344/17-ways-to-stop-pipetting-errors-ruining-your-experiments/
http://www.gilson.com/Resources/Gilson%20Guide%20To%20Pipetting%20Third%20Edition.pdf
Teaching: https://www.cpet.ufl.edu/wp-content/uploads/2012/10/Pipetting-by-Design-lesson-plan-6_2012.pdf
