The Holding Pipet

1. Connect a Leitz instrument tube to an Agla micrometer syringe with 1 M Tygon tubing. Fill the system with light paraffin oil making sure that all the air bubbles have been excluded. Position the Agla micrometer syringe in a clamp stand in a convenient position close to the left-hand manipulator.

2. Fill the holding pipet with Flourinert using a needle (26-gage, 5 cm) attached to a 1-mL disposable syringe. Insert the holding pipet into the oil-filled Leitz instrument tube, and tighten the ring to hold it in place.

3. Clamp the instrument tube into the instrument tube holder of the left-hand micromanipulator (if one is right-handed). Adjust the Agla micrometer until fluid stops flowing out of the holding pipet. Do not allow air to flow back into the holding pipet.

4. Position the holding pipet 2 cm above the center of the microinjection chamber making the necessary adjustments, such that the tip of the holding pipet is horizontal and straight. Monitoring under the 4x magnification, use the fine controls of the micromanipulator to lower the holding pipet into the injection chamber. Adjust the manipulator such that the tip of the holding pipet is just above the floor of the chamber. The holding pipet should move freely in the horizontal plane throughout the field of vision and not catch on the floor of the chamber.

5. Using the Agla micrometer, draw M2 medium into the holding pipet until the meniscus between the M2 and Fluorinert is just at the shoulder of the holding pipet.

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