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Transfer the straw from the
LN2 storage freezer to a smaller container of LN2.
Using forceps, grasp the straw near
the label and hold it at room temperature for 40 seconds, then
submerge the straw in room temperature water until the ice
Wipe straw dry.
Holding the straw firmly, cut off the
seal and cut through the PVA plug, leaving about half of the cotton
plug in place.
Using a metal rod as a plunger, push
against the remaining portion of the cotton plug and expel the
entire liquid contents of the straw into a 35 mm culture
dish. Do not let the plug drop into the dish.
Wait 5 minutes. The embryos will
Transfer the embryos to a drop of
M2. They will rapidly take up water and assume a normal
appearance. Wait 5 minutes.
Wash the embryos in a fresh dish of
M2. Wash embryos through 8 to 10 drops of media. Place
embryos in drops of KSOM overlayed with mineral oil.
Transfer the embryos either into the
oviducts of a 0.5 dpc pseudopregnant mouse or culture to the
blastocyst stage in KSOM and transfer to the uterus of a 3 dpc