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RESEARCH DIVISION Laboratory Manual

 


 

Preparation of Microinjection Pipettes

Injection and Holding Pipettes

The glass capillary tubing used should be thin walled, borosilicate glass without a fibre.

e.g. Clark Electromedical Instruments

- GC 100T - 15 (= 15 cm)
- GC 100T - 10 (= 10 cm)
- OUTSIDE DIAMETER: 1.0mm
- INSIDE DIAMETER: 0.78mm
- LENGTH: 150mm

Australian Agents:

SDR Clinical Technology, 213 Eastern Valley Way, Middle Cove NSW 2068 (02) 958 2688

Glass should be acid washed:-

  1. Place glass in a 100 ml measuring cylinder, containing 1:1 v/v Sulphuric acid, Nitric acid, soak overnight. This is done in fume hood, make sure gloves and goggles are worn.
  2. Pour acid into another cylinder, this can be reused.
  3. Wash glass at least 10x with Milli Q H20 then 3x with Travenol H20. Drain well
  4. Cover cylinder with parafilm, poke holes in it using a needle and place glass in 60C oven until dry. This takes a few days.

Pipette pulling:-

- Flamming Brown micropipette puller Model P-87
- Sutter Instrument Co. USA
- Using Program 6 (VH)
- Heat = 915 Pull = 20 Vel = 90 Time = 120 Pressure = 500

Injection pipettes:-

Injection pipettes should have an internal diameter only marginally larger than the stem cells (18-20 um) and a sharp point which allows the needle to penetrate cleanly in the blastocoel cavity.

The glass is cut by hand under a stereo dissecting microscope, at a magnification of X50, using a sharp scalpel blade and a large glass petridish filled with silicon. (Dow-corning Silgard). The glass will usually break with a sharp bevelled end when touched with the edge of the scalpel blade. The resultant pipette should have a relatively long section at the appropriate dimater (18-20 m).

Holding pipettes

Holding pipettes are blunt flame-polished pipettes through which suction is applied to immobilize the blastocyst. The same piece of glass is cut, only further up the shaft ~200 m. This is cut by holding the shaft of glass on your forefinger with your thumb and scored with a diamond pencil then broken 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 um. (1-2 divisions).

 

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998