There’s lots to say about pipetting little amounts!
Firstly, if you don’t have to, don’t! Avoid it altogether!
- pre-dilute concentrated solutions so you can pipet larger volumes
- use mastermixes when possible: bit.ly/mastermi... ; • Mastering master mixes...
- “never” try to pipet less than 0.5 μL
Don't pipet into thin air!
- pipet into liquid or onto wall of container so surface tension can help pull it all out
Check to make sure the liquid's really there!
- look at the tip to make sure the liquid got sucked up (without bubbles) and all got dispensed out
- if there is still liquid in the tip, go back in, pull up until liquid reaches the trapped liquid and use it to drag that liquid back down
- make sure the liquid line is consistent each time
Alter angles to achieve accuracy
- aspirate (suck up) vertically (and slowly)
- avoids capillary action causing you to suck up too much
- dispense (slowly) at a 45° angle
- helps it all gets out
Make sure to practice proper thumbmanship
- only go to first stop when aspirating (don’t push thumb all the way down at this point)
- go to second stop (all the way down) when dispensing
- pause (with thumb pushed down) before pulling out
- keep thumb pushed down when you pull out
- pull out slowly, making sure all the liquid gets pulled out before you release your thumb
- if there is still liquid in the tip, go back in, pull up until liquid reaches the trapped liquid and use it to drag that liquid back down
Stay just below the surface
- avoid liquid sticking to outside of tip (look to make sure as well)
Use the smallest possible pipet
- use P2 when possible, especially for volumes under 1 uL
Use low-binding tips
Call that calibrator!
- make sure the pipets are regularly calibrated to ensure accuracy
To err is human, and/or humidity
- differences in air temperature inside the pipet tip can alter the pipetted volume; if the air is warmer inside, liquid will evaporate and decrease the actual liquid volume
- avoid warming up the air in the pipet
- don’t hold on to the pipet while you’re waiting around
- pre-wet the tip?
- pipet up & down several times first
- eppendorf recommends this, but anecdotally I’ve had trouble with this due to sample getting stuck on in tip and altering volumes, etc.
Practice
- pipet drops of water onto Parafilm on an analytical scale
- water has a density of 1 g/mL, so 1 mg/μL, so if you pipet 1 μL, it should weight 1 mg. each time
blog version: bit.ly/pipetti...
resources:
Eppendorf, “How to Pipette Small Volumes with Handheld Manual Pipettes”, 09/19/18 handling-solut...
Gilson, “Pipetting Small Volumes: 4 Tips for Improving Your Technique” www.gilson.com...
ThermoFisher Scientific, “Proper Pipetting Techniques” www.thermofish...
more pipetting tips: • Pipetting tips
more practical lab tips & tricks: bit.ly/lab_tri... & • Practical lab tips & t...
more on accuracy vs. precision: bit.ly/accurac... ; RU-vid: • Precision vs accuracy
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbioc...
22 авг 2024
