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
Biopsies/milking - 0.9-1.5 ml IP
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
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
Thousand Oaks, CA