Suction/injection pipettes

doi:10.1101/pdb.rec12094

Suction/injection pipettes

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MATERIALS

Reagents

Fluoric acid (2%)

Equipment

Abrasive film, 0.3-μm (e.g., Thomas Scientific 6775E54)

Capillary tubes, borosilicate glass, 10-cm length, 1-mm OD, 0.5-mm ID (e.g., Clark or equivalent)

Container (clean, airtight)

Microforge, equipped with micrometer scale eyepiece (e.g., deFonbrune; Alcatel or equivalent)

Micropipette puller (e.g., Campden Instruments Ltd. or equivalent)

Pipette grindstone

Rubber tubing

Syringe, 10-mL

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METHOD

1. Switch on the micropipette puller. Program with the following recommended settings:
1. Prepull heating time: 048
2. Initial pull force: 958
3. Main pull force: 395
4. Main pull delay: 062
5. Heater control: 140
2. Place the capillary tube in the micropipette puller according to the manufacturer instructions. Press “Load” and “Pull” to obtain a pulled capillary.
3. Clamp the pulled capillary into the microforge.
4. Turn on the microforge. Guide the pulled capillary horizontally relative to the heating filament (i.e., at the level of the small glass ball) and slightly below it.
5. Using the 20X objective and the micrometer scale eyepiece, focus on a region with a 7-μm OD.
6. Heat the filament to ~60°C.
7. Move the pulled capillary toward the heating filament. When the capillary reaches the small glass ball, stop heating. Raise the pulled capillary quickly.

This should cut the pulled capillary; verify that the capillary is cut straight.
8. Bevel the capillary tip with a grindstone and abrasive film to produce a tip with an OD of 7-10 μm.
9. Using a syringe with attached rubber tubing, rinse the beveled injection capillary for a few seconds with 2% fluoric acid to remove the glass dust. Rinse well with water.
10. Guide the pulled capillary vertically relative to the heating filament. Move the heating filament close to the capillary (e.g., 2-5 mm from the tip of the pipette).
11. Carefully heat the filament to ~70°C until the capillary begins to bend. Stop heating when the desired angle is obtained.

The angle will vary according to the type of micromanipulator used.
12. Store suction/injection pipettes in a clean airtight container until use.