Reagents, supplies, and equipment:
- M2 medium (Millipore MR-015P-5D)
- KSOM medium (Millipore MR-020P-5D)
- 35 mm culture dishes
- Paper clip for plunger
- Scissors
- LN2
- Long forceps
- Room temperature water bath
Thawing 2-cell stage embryos in straws:
- 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 disappears.
- 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 shrink considerably.
- 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 recipient.