- M2 medium (Millipore MR-015P-5D)
- KSOM medium (Millipore MR-020P-5D)
- paper clip for plunger
- 35 mm culture dishes
- long forceps
Thaw embryos in straws:
- Transfer the straw from the LN2 storage freezer to a
smaller container of LN2.
- Using forceps, grasp straw near the label and hold it in the
air for 40 seconds, then submerge in room temperature water until
the ice disappears.
- Wipe straw dry.
- Holding firmly, cut off seal and cut through PVA plug, leaving
about half of the cotton plug in place to act as a plunger.
- Using a metal rod, expel the entire liquid contents of the
straw into a 35 mm culture dish. Do not let the plug drop into the
- Wait 5 minutes. The embryos will shrink
- Transfer the embryos to a drop of M2. They will rapidly
take up water and assume a normal appearance. Wait 5
- Wash the embryos in a fresh dish of M2 (wash through several
drops of media if from a contaminated source - 10 times total) and
then, either transfer to oviducts of a 0.5 dpc pseudopregnant
mouse, or culture to blastocyst stage in KSOM and transfer to
uterus of a 3 dpc recipient.
Thawing 8 Cell Embryos in Vials (Frozen
using JAX slow freeze protocol):
- Remove cryo tubes from LN2 storage and place on the
bench at room temperature until thawed, ~ 12-15 minutes.
- When completely thawed, slowly add 0.8 ml of PBS to the cryo
vial in a dropwise fashion to dilute the DMSO.
- The contents of the cryo tube are withdrawn by means of a
Selectapette (Clay Adams).
- Wash embryos once in KSOM medium before putting into
*Additional washes (up to 10) may be necessary when cleaning up