A02 Pipetting

Purpose

To use an automatic pipettor.

Procedure for Automatic Pipettors:

Observe the pipetting procedure. Review important points as needed.

Reusable pipets are cleaned, plugged with cotton, sorted into cans by size, and autoclaved. Cans of sterile pipets are placed in the hood. When in use, the covers may be removed from the cans. They are covered at other times. Cans of plugged, disposable Pasteur pipets are also placed in the hood.
Select the can of appropriately-sized pipets. Tilt the can. Gently shake the can so that the mouths of the pipets become accessible. Use forceps to remove one pipet. If one pipet shakes clear of the others, it may be grasped by fingers.
Insert the mouth of the pipet snugly into the pipettor. Press the top button to draw fluid into the pipet. Press the bottom button to deliver fluid from the pipet. Uncover the fluid to be pipetted. Immerse only the tip of the pipet into the fluid to be pipetted and withdraw the correct-sized sample. Remove the filled pipet and cover the fluid source. Uncover the receiving vessel. Place the tip of the pipet into the receiving vessel and deliver the fluid. Remove the pipet and cover the receiving vessel.
Remove the pipet from the pipettor. In some laboratories, the cotton plugs are removed at this point. Use forceps to grasp the plug. Discard the cotton plug with the trash to be autoclaved. Place the pipet in a receiver containing detergent and disinfectant.
Pipetting is the most commonly used technique at the hood. It must be learned well so that experiments are performed speedily while aseptic conditions are maintained.

Using Micropipettors:

Observe the pipetting procedure. Review important points as needed.

Adjust the volume to be delivered by setting the control knobs on the side of the handle.
Disposable pipet tips are autoclaved to sterilize them and to inactivate contaminating enzymes.
Disposable tips are kept covered to maintain sterile conditions. To remove a tip, lift the cover and insert the tip of the pippettor into one of the tips. Remove, and close the lid.
Disposable tips are removed from the pipettor by holding it over an appropriate waste container and pressing the tip ejector button.
To fill the pipet, depress the top button. Immerse the tip beneath the surface of the liquid. Slowly release the button. To deliver a measured volume of liquid, depress the button. Depressing the button expels fluid, be that air or liquid. Releasing the button takes in fluid, be that air or liquid.
The pipettor may be used to mix liquids by alternately depressing and releasing the button.
The pipet tip may be used to push droplets separated from the liquid bulk back into the bulk. This is often necessary for radioactively labelled reagents as supplied by the manufacturer. A variety of pipet styles are available, including some which refill from a reservoir and, therefore, cut the number of operations nearly in half.
Maintain pipettors in good working order. Check calibrations frequently.
Use separate pipettors for handling radioactive materials.