Embryo Manipulation Pipettes (Flushers etc.)
Glass used for making transfer pipettes :
Agent in Australia :
Before use, the capillary glasss should be acid washed (see protocol "Preparation of Pipettes").
Setting up the Microburner
Transfer pipettes are pulled by hand over a small flame from a microburner. A microburner can be set up using rubber tubing, a retort stand, a 19 gauge needle and an adustable screw clamp. Attach the needle to the end of the tubing to give an airtight seal around the needle base. Using the retort stand clamps, set the tubing up so that it is stable with the needle pointing upwards. Place the adjustable screw clamp around the tubing (below the retort stand clamps) and tighten fully. Connect the other end of the tubing to a gas outlet and the microburner is ready for use. The adjustable screw clamp is used to increase/decrease the amount of gas leaving the needle and thereby controlling the size of the flame.
Pulling Transfer Pipettes
The flame on the microburner should be set low (about 6mm high). Adjust the height of the needle of the microburner (by moving the retort stand clamps) so that you can rest your elbows on the bench whilst working with the glass in the flame. This is quite important if straight pipettes are to be pulled. Resting your elbows on the bench, take a piece of glass and hold both ends between thumbs and two forefingers. Hold the middle of the glass over the flame and roll gently between fingers, keeping the glasss in the flame at all times. After a few seconds, the glass being heated should begin to glow red and become flexible. In one smooth movement, bring the glass forward (out of the flame) and pull the two ends away from each other, hopefully to produce a thin, even length of glass as shown below.
Snap glass with fingers and store in large petri dish. The external diameter must be 190-230um.
Before use the pipettes are cut to a length at which they are comfortable to use, i.e. ~ 2 cm length of fine-pulled glass. The glass is scored at the desired length using a diamond pencil. The scored site should snap when pressure is applied to it, giving a clean square break. The edges of this break are still quite sharp and they may be bluntened softly by flame polishing. This is not really necessary for embryo manipulation but should always be performed on pipettes used for embryo transfer into pseudopregnant mice.
Flame Polishing Transfer Pipettes for Blastocyst Transfer
Transfer pipettes used for blastocyst transfer need to have the end polished to give a rounded smooth tip which is inserted into the uterus.
Transfer pipettes which are used to transfer blastocysts from injection should be flame-polished, non-siliconized glass and must have an internal diameter of 100um-110um.
Transfer pipettes should be flame-polished, siliconized glass with an internal diameter of 120-150um as this is the size which most aggregated blasts will expand to. ALWAYS MEASURE THE DIAMETER OF AGGREGATED BLASTOCYSTS BEFORE YOU SELECT A TRANSFER PIPETTE This will ensure that you have a pipette which is wide enough to accomodate the largest expanded blastocysts from your petri dish and they will not be exposed to the additional trauma of collapse as a result of a small pipette.
The same glass as that used for preparing transfer pipettes.
Flushers are hand-pulled over the small flame (a bit longer than for transfer pipettes) of a microburner
See protocol on morula flushing for information on (see protocol on Transfer Pipettes). The flushers are pulled by holding the glass at both ends and heating the glass in the flame whilst pulling steadily but firmly on both ends. The glass will melt and pull away quickly. The result should be a short sharp point, as shown below.
The flushers should be pulled at both ends so that one end is sharp for flushing, and the other end is tapered and fits smoothly into the flushing tubing. Two flushers can be obtained from one 15cm length of glass by pulling once in the middle and once at each end.set up for flushing apparatus.
|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: firstname.lastname@example.org |
David Bowtell PMCI October 1998