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Avertin Usage

I. Components

a. Avertin (2,2,2 tribromoethanol, Aldrich T4,840.2 or eq.)
b. Tert-amyl alcohol

II. Stock solution (1.6 g/ml)
Add 15.5 ml T-amyl alcohol to 25 g. avertin in dark bottle (the bottle that the avertin is shipped in works great). Stir on magnetic stirrer until the avertin is dissolved (about 12 hours). Avertin stock is light sensitive and hydroscopic. Keep in dark bottle at RT (If the solution is kept at 4° F. the avertin will "freeze" out, necessitating re-dissolving the avertin), away from light and tightly sealed. Do not leave the bottle open longer than necessary.

III. Working solution (20 mg/ml)
Mix 0.5 ml avertin stock solution and 39.5 ml normal saline in glass vessel (graduate cylinder works great). Seal container with parafilm, wrap in foil to exclude light and stir on magnetic stirrer for about 12 hours or until dissolved. Filter sterilize through 0.2 micron filter and store at 4° C. It can be aliquoted into ~5 ml lots in foil wrapped, sterile serum vials or kept in a dark, capped bottle at 4° C.

IV. Dosages (0.4-0.6 mg/gm)

a. Embryo transfers - 0.45-0.75 ml IP

b. Biopsies/milking - 0.9-1.5 ml IP

It will take about five minutes for the mouse to become fully anesthetized (lack of toe pinch reflex). An additional 0.1-0.2 ml can be given to effect. The mouse will remain anesthetized for approximately 15-20 minutes and recover within 30-60 minutes. Keep mouse warmed during recovery. I place the cage on an electric heating pad set to the lowest temperature. Note that the effective dosage is dependent upon the weight of the mouse. Older, fatter or lactating mice will need more avertin to become fully anesthetized. It is difficult to over-anesthetize (kill) the mouse even at higher dosages.

V. Shelf life
The stock avertin solution is quite stable at room temperature. I have used it for up to a year after preparation with no problems. Yellowing of the solution indicates oxidation products (toxic) and the stock must be replaced.

Working solutions must be kept refrigerated in a dark bottle until use and should be replaced at least every month.

This preparation method was used in Jeff Rosen's lab at Baylor College of Medicine in the late '80s. It has always worked well for me and I can't recall ever losing a mouse due to over-administration. No ill effects have ever been seen in the avertin-anesthetized mice, either post-op or during pregnancy and lactation. You do need to watch for an inadvertent IV injection, which will kill the mouse within minutes.

Each preparation of working solution should be checked prior before use to determine the best dosage range and to verify that it is effective. Make sure that the test mice are effectively anesthetized and recover completely before use in any transgenic manipulations.

DISCLAIMER: Neither Amgen Inc. nor myself take responsibility for the efficacy or safety of this preparation nor responsibility for any damages or liability for its use.

Above information taken with permission from:
Joe Anderson, MS
Transgenic Services
Amgen, Inc.
Thousand Oaks, CA
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