Pipetting techniques and mistakes guide.
1. Wrong immersion depth of the tip
The depth of immersion of the tip can improve the accuracy by up to 5%. The tip should be submerged 1 or 2 mm in the microvolume pipettes and up to 3 or 6 mm in the large volume pipettes, depending on the size of the tip. If the tip dips too much, the gas volume of the tip is compressed, and it takes too much volume.
2. Incorrect pipetting angle
The immersion angle of the tip in the liquid should be as vertical as possible and should not deviate more than 20 degrees from the vertical angle. More pronounced angle causes too much liquid to enter the tip, which leads to inaccurate aspirations. For example, at an angle of 30 degrees to the vertical, you can take up to 0.7 % excess fluid.
3. Irregular dosage
To achieve superior accuracy and reproduction between samples, be sure to dose every last drop of the sample, even on the end of the tip. For most applications, dosing with the end of the tip resting against the wall of the container is recommended, and thus reducing the amount of sample remaining at the tip. This technique can increase accuracy by 1% or more.
4. Pre-rinse failure
The dosing of the liquid from a pipette leaves a liquid coating on the tip, which causes the dispensed volume to be slightly lower than it should be. Pre-rinsing a new tip a couple of times, at least, with the liquid that is going to be used, will serve to condition the inside of the tip.
5. Irregular rhythm when pippeting
Use a steady rhythm in pipetting between samples. Avoid going fast or performing quick operations and keep pace for each step of the pipetting cycle.
6. Do not assess the viscosity of the sampled
It is important to note the physical properties of the sample, since they directly affect the volume dispensed. In the case of dense and / or viscous samples, which tend to adhere to the surface of the tip, or on the contrary, the more volatile samples, which will be dispensed more quickly and will tend to evaporate.
Some of the ways to minimize these effects are through the use of ultra-low retention pipette tips, which contain a hydrophobic plastic additive that prevents the liquid from adhering to the inside of the tip.
7. Dispende liquid reagents too quickly
Pipetting too quickly increases the probability of making mistakes when dispensing the correct volumes, as well as favoring the contamination of pipettes, among others.
8. Pipette different samples with the same tip
If the same tip is used to extract and dispense a sample and then immediately pipette another sample without changing it, contamination of the samples may be induced.
9. Not performing proper maintenance of the pipettes
Pipettes can lead to error in the measurement of volumes or cause contamination if not maintained correctly. Daily cleaning with 70% ethanol is essential, as well as the use of different pipettes for general tests and for more specific tests.
10. Do not calibrate the pipettes within the established deadlines
It is recommended to calibrate the pipettes at least once a year, and ideally every 3 months, to ensure that the measurements made with them are accurate.
11. Aspirate air
Although it seems simple, it is a common error to pay attention to, since in addition to pipetting an inaccurate volume, when sucking air the liquid can enter the neck of the pipette.
12. Storing the pipettes horizontally
Pipettes should be stored in an upright position, preferably on supports specially designed for them. This prevents liquids that may have entered the neck of the pipette from escaping and causing contamination and / or corrosion phenomena.
13. Do not use the appropriate tips for the pipette
Pipettes should be stored in an upright position, preferably on supports specially designed for them. This prevents liquids that may have entered the neck of the pipette from escaping and causing contamination and / or corrosion phenomena.
14. Do not pre-moisten the tips
By submerging the tips the humidity inside them increases, thus minimizing the evaporation of the solution. This practice is usually recommended for volumes greater than 10μL.
15. Do not take into account the ambient temperature and/or the temperatura of the sample
The calibration of the pipettes is usually done at room temperature, so that if you work at significantly lower or higher temperatures, the measurement will not be accurate. The temperature of the samples can also cause the volumes dispensed to vary.
