Preparing Micropipettes for Oocyte Injection

Pulling glass pipettes for microinjection and TEVC

1. Turn on the pipette puller using the power switch on the left side. Determine the program you need by checking the program index on the cover. Select the program by entering its number and pressing ENTER.

2. Locate an 8-inch glass capillary tube for microinjection or a 4-inch glass capillary tube for TEVC. Clean the tube with a Kimwipe, focusing on the middle, where the break will be made.

3. Slide the glass tube in the groove on the right puller bar and allow it to go off the bar (~1 in) toward the heating filament, which is the metal ring in the center. Tighten the knob just enough that you can still slide the capillary along the groove while the capillary is still secured in it. Do not loosen the knobs so much that the tubing falls.

4. Press the metal flap above the puller to release and slide the puller toward the middle until it stops. Hold it in place. Allow the capillary to go through the heating filament.

5. Using the same hand, repeat the same process for the left puller and push the capillary into the left groove. Do not release the puller bars as they will recoil back. Tighten the left knob enough to secure the capillary but to allow the capillary to slide in the groove.

6. With your other hand, align the middle of the capillary with the heating filament. There should be equal lengths of the capillary on either side of the heating filament.

7. Tighten both knobs all the way and release the puller bars. Close the lid and press PULL to run the program.

8. When the process is complete, remove both capillaries (now called pipettes) by holding the part over the top indentation in either puller bar. Loosen the knob the release the micropipettes. Place them immediately in the appropriate micropipette container for safe keeping. BE CAREFUL NOT TO BRUSH THE TIP ON ANYTHING AS THIS WILL BREAK THE TIP.

Breaking microinjection pipette tips for injection

Microinjection pipettes made in the pipette puller have an extremely narrow opening. These pipettes are used for injection into oocytes, which requires the passage of solutions through the tip. Before new pipettes can be used for injection, the tips must be broken to obtain a width of 9-15 um.

1. Obtain an unbroken pipette from the UNBROKEN plastic dish and a Kimwipe. Set the Kimwipe on a hard surface (i.e. table) and gently drag the tip on the Kimwipe. The goal is to be able to break the tip to an acceptable size. The pipette dragging technique is highly variable among different people and only practice will allow you to learn how far to drag the pipette for or how much force should be applied. You may drag in a single long stroke or in multiple shorter ones.

2. When done, the size of the tip has to be verified with the microforge. Being careful not to touch the heating wire, place the micropipette tip down in the holding mechanism of the microforge by sliding the micropipette under the metal clip and into the groove.

3. Make sure that the micropipette is far down enough to be seen with the objective lens. You can either turn the right knob on the mechanism to lower it or you may manually push the micropipette down.

4. Look down the microforge and bring the micropipette tip into focus on high magnification by turning the fine adjustment knob ONLY. The lightest turn of the coarse adjustment knob can push the objective lens into the heating filament and micropipette, damaging the lens and other parts.

NOTE: If you’re having trouble locating the micropipette, switch to low magnification, focus the micropipette, and then switch back to high magnification. You may also try to move the micropipette up/down, left/right with the holding mechanism adjustment knobs to help you find it.

5. The goal is to have the opening of the tip be 3 to 5 units (~9-15 um) on the ruler while on the higher magnification. If the tip is the correct width, skip to step 7. If the tip is too large, dispose of it in the broken glass container.

6. If the tip is too small, carefully remove the micropipette from the holder and drag the tip on a Kimwipe again. Check the new width and repeat as needed.

7. Once the tip is the correct width and if the edges on the tip are jagged, use the knobs to center the tip directly over the heating wire. You may need to do this on lower magnification to view the micropipette and the wire.

8. Step on the foot pedal to allow the wire to heat and fire polish the micropipette. Fire polish in 2-second bursts and repeat as needed. You will rarely have to fire polish for an extended time.

9. Once the micropipette is done, place it in the plastic dish for broken micropipettes. Note that the micropipettes are sorted in order of increasing tip diameter. Place your micropipette accordingly.