Using a pipette the correct way can lead to better data and more reliable results. Here are a few of the ways you can control variation between wells.
Pre-wet your pipette
For all volumes over 10μL, aspirate and dispense at least 3 times to increase humidity within the tip and reduce the amount of variation by evaporation.
Visually look at amounts of liquid in your pipette
You may be able to tell visually if there is any variation between volumes of liquid in each tip of your multichannel pipette. (See Figure 1)
Loose tips can cause variation between the volumes of each well and lead to skewed data. (See Figure 2)
Touch down once
Avoid sliding pipette tips down the side of a well. This can lead to residue clinging to the sides of the wells which leads to background in the well. Instead, just touch your tips down one time in the bottom corner of the well and dispense liquid. Sliding tips in the bottom of the wells can also smear spots leading to unclear results.
Minimize the amount of times you have to pipette
Pipette the fewest number of times possible. Choose a pipette volume that will minimize the number of times pipetting into a well. The fewer times you have to pipette, the less chance there is for error.
Minimize bubbles as much as possible
Try not to blow any air into your wells when dispensing the liquid. This is especially critical when adding substrate before imaging. (See Figure 3)
Pipette large volumes over small
Large volumes are easier to pipette accurately. Pipetting 50μL is more likely to be accurate than pipetting 5μL.
10% rule for accuracy of volumes
Pipettes are less accurate at minimum and maximum settings. Avoid using pipettes for volumes less than 10% of the maximum volume.
Stick to one way of pipetting and do not switch methods during an assay. There are 2 popular ways to pipette:
-standard or forward pipetting
Depress plunger to first stop and aspirate. Then dispense to first stop, discard excess.
Depress plunger to second stop and aspirate. Then dispense to first stop/ discard excess (this is only if you have enough sample that discarding some liquid is not troublesome).