Pipetting is a necessary skill for setting up a qPCR reaction. This article will provide an introduction to using a pipette and some common practices.
Parts of a pipette
The Plunger of the pipette can be pressed down and released up to pick up solution. The plunger can be pressed down with the thumb to one level, and then further to a second level. The first level is utilized for initial volume pick up and release, while the second level is used to expel any lingering solution.
The Volume Adjustment can be twisted to change the Volume Readout of how much volume of solution will be transferred. The Volume Lock must be slid upwards to change volume and then can be slid downwards to prevent accidental adjustment during pipetting.
The Tip Eject Button is pressed down with the thumb when pipetting is complete and the pipette tip needs to be removed. Only press this button when holding the pipette over a waste container because the tip will come off rapidly. This mechanism allows the tip to be removed without physical contact.
The Tip Attachment is the region of the pipette that is pressed into a pipette tip in order to attach a tip.
How to Pipette
- Select the appropriate pipette depending on the volume being transferred. The desired volume must fall within the range allowed by the pipette. Pipetting a volume outside of the pipette’s range can damage the pipette and should be avoided at all times.
- Slide the volume lock upwards to unlock the adjustment. Set the volume readout to the required volume using the volume adjustment. Slide the volume lock downwards to lock in the desired volume.
- To attach a filtered pipette tip, firmly press the tip attachment portion of the pipette directly down into a tip, then lift up the pipette—the tip will be attached.
- Push the plunger down to the first level. Insert the pipette tip into the tube from which solution is to be removed.
- With the tip submerged in the solution, slowly release the plunger. This will draw liquid up into the pipette tip. Ensure that the tip is completely submerged to avoid air bubbles. Remove the pipette tip from the tube.
- Insert the pipette tip into the tube where the solution is to be transfered. Push the plunger down to the first level, then push down (and hold) at the second level to release any final liquid into the tube. Lightly press the tip against the inside of the tube to transfer any remaining liquid droplets.
- Still holding the plunger down at the second level, remove the pipette tip from the tube. Make sure all liquid has been released from the pipette tip. Release the plunger completely.
- Use the tip eject button to remove the tip into waste without having to touch the tip.
Use filtered pipette tips to prevent potential contamination of your pipettes. The filter acts as a barrier to prevent potential contaminants from being taken up into your pipette. Pipettes should be wiped down regularly with aqueous detergent, such as 5% bleach, to prevent contamination. Always wear gloves when handling pipettes and store pipettes in a designated location.
Change pipette tips as necessary. If you are pipetting one solution into multiple empty tubes, the same pipette tip can be used, but make sure that the tip doesn’t touch any surface or other solution. If this happens, eject the tip and get a new one. Always use a new tip for each solution to avoid cross contamination.
When pipetting multiple solutions into one tube, pipette the larger volumes first. Then add the smaller volumes to the larger volume. This will help prevent pipetting error and avoid loss when one volume is a very small amount.
“Pipetting up and down” is the technique of using a pipette to mix the solution in a tube. To do this, press the plunger to the first level, then place the tip into the solution to be mixed. Slowly alternate the plunger between the released state and first level between 5-10 times. Ensure the tip is completely submerged the entire time to avoid air bubbles. After pipetting up and down, press and hold the plunger down to the second level before removing the tip from the tube so that all solution is released from the pipette tip.
Viscous solutions such as saliva may take slightly longer to be pulled up into the pipette tip. When pipetting a viscous solution, be sure to release the plunger slowly to allow adequate time for the solution to be drawn up into the pipette tip. Allow the pipette tip to remain submerged in the liquid for a few seconds after drawing up the solution to ensure full uptake.