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    • CommentAuthorkalayman
    • CommentTimeMar 30th 2010 edited
     

    This protocol explains how to: make a microinjection mold out of agarose. Embryos are put into the grooves created by the mold, which keep them stable during microinjections.

    Adapted from: http://site.adaptivesciencetools.com/Images/moldinstructions.pdf (visit website for pictures of setup)

    1. Boil (by using microwave) 3% agar into solution. DO NOT PLACE THE MOLD INTO BOILING AGAROSE DIRECTLY OUT OF THE MICROWAVE. Extreme heat causes warping of mold.

    2. Cool agarose to 45ºC on bench top. Use a digital thermometer to monitor the temperature.

    3. Attach a tabbed piece of tape to the backside of the dried mold. This acts as
    a handle to control the mold plate as it enters the agarose.

    4. Pour the cooled agarose (45ºC) into a Petri dish.

    5. Allow one end of the mold to contact liquid, then lay the mold on top of the
    liquid in one smooth motion. This “floating action” eliminates air bubbles.

    6.  Once the mold is in the liquid agarose, allow it to solidify at room
    temperature.

    7. Place the agarose petri dish that has firmed to an opaque color into a 4ºC
    refrigerator for approximately 30 minutes.

    8. Slip a spatula gently under the perimeter of the mold to assist separation
    from the agarose.

    9. Slowly pop the mold out. If done properly, the mold should release cleanly.

    10. The gel injection plate you just made is reusable. Flood the impression with
    same liquid used to make the gel, cover the dish, and refrigerate.