The Microinjection Pipet

1. Connect a Leitz instrument tube holder via 1 m of Tygon tubing to either a 50-mL syringe with ground-glass plunger (lubricated with liquid paraffin) or an automatic injection system. The system is air-filled.

2. Backfill a freshly drawn microinjection pipet with DNA. Place the end distal to the tip into the DNA solution, allow the liquid to ride into the tip by capillary action and withdraw when liquid can be seen in the tip. Avoid contaminating the DNA stock solution, e.g., with glove powder or enzymes from the exposed hand.

3. Assemble the microinjection pipet into the instrument tube. Clamp the instrument tube onto the instrument tube holder of the micromanipulator.

4. Position the microinjection pipet until the tip is 2 cm above the center of the microinjection chamber and 15-20° to the horizontal. Using the fine vertical control of the micromanipulator, lower the tip into the injection chamber until it is just above the chamber floor. Monitor the position of the tip throughout using the 4X objective.

5. Using the horizontal controls of the micromanipulators, bring both the injection pipet and the holding pipet to the center of the field of view.

6. Switch to the 40x Normarski objective, and focus on the HP. Using the fine controls of the micromanipulator, bring the tip of the microinjection pipet into the same focal plane as the holding pipet. The microinjection pipet should move freely in the horizontal plane when operated by the joystick control.

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