>

Thaw Embryos in Straws

Reagent and supplies

ReagentSourceCatalog Number
EmbryoMax M2 mediumSigmaMR-015-D
EmbryoMax KSOM AdvancedSigmaMR-101-D
EmbryoMax mineral oilSigmaES-005-C
35 mm tissue culture dishesFisher0877230
60 mm tissue culture dishesFisher0877231
Paper clip for plunger
ScissorsFisher08950
Long forcepsFisher10316C
Beaker with room temperature water

Prepare culture dishes (one set per straw thawed)

  1. One 35 mm dish for depositing thawed embryos in.
  2. Prepare one 35 mm dish containing a 50 μl drop of M2 for the rehydration step.
  3. Prepare one 60 mm dish containing 8-10 drops of 50 μl M2 medium for embryo washing.
  4. Prepare one dish with KSOM drops overlaid with mineral oil for embryo culture.

Thawing embryos in straws

  1. Transfer straw from LN2 storage to a small LN2 container.
  2. Grasp the straw near the label and hold it at room temperature for 40 seconds, then immerse in room temperature water until the ice disappears.
  3. Wipe straw dry.
  4. Cut off the seal and cut through the PVA plug, leaving half of the cotton plug.
  5. Using a metal rod as a plunger, push against the remaining portion of the cotton plug and expel contents into a 35 mm culture dish; avoid dropping the plug.
  6. Wait 5 minutes for embryos to shrink.
  7. Transfer the embryos to M2; allow rehydration for 5 minutes.
  8. Wash through 8-10 drops M2.
  9. Place embryos in drops of KSOM overlaid with mineral oil.
  10. Embryos may be cryopreserved, cultured, or transferred to 0.5 dpc recipients.