Microinjection Pipette Pulling Protocol

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Microinjection Pipette Pulling Protocol

Date Added: March 02, 2008 09:50:03 PM

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Category: Stem Cell Protocols

Pipette Pulling Protocol

For pipette pulling, use the Flaming Brown micropipette puller Model P-87 (Sutter Instrument Co.), using Program 6 (VH).

Set the following parameters for the machine:

Heat = 915
Pull = 20
Vel = 90
Time = 120
Pressure = 500

After pulling the glass pipettes, they are then cut by hand under a stereo dissecting microscope, at a magnification of 50X, using a sharp scalpel blade and a large glass petri-dish filled with Silicon (Dow-Corning Silgard). The glass will usually break with a sharp beveled end when touched with the edge of the scalpel blade.

The resultant pipette should have a relatively long section at the appropriate diameter (18 to 20 μm).

Holding pipettes are blunt flame-polished pipettes through which suction is applied to immobilize the blastocyst. The glass is prepared in the same manner as the injection pipettes but are cut approximately 200 μm further up the shaft. This is done by holding the shaft of glass on your forefinger with your thumb and scoring it with a diamond pencil then breaking it by flicking the surplus off the top. A clean neat break is essential, otherwise the blastocyst does not sit properly on the end and comes off during the injection process. The end of the pipette is then flame polished by holding it in a microburner flame until the internal diameter is reduced to 20 μm (1 to 2 divisions).

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