Appendix 9: Vitelline Envelope Removal

Eggs obtained by Method I are quite fragile and must be handled carefully (minimize centrifugation, pipetting, etc.). We have not tested Method II, but expect that the eggs should be equally fragile after vitelline envelope removal by either method.

Method I (Eckberg and Anderson, 1985; Polk et al., 1987)

  1. Wash eggs in at least 10 vol CaFASW.
  2. Handle eggs very gently from this point on.
  3. Suspend eggs 30-60 s. in an equal volume of: 500 mM sucrose, 125 mM EDTA, pH 8.0.
  4. Add at least 5 volumes of CaFASW.
  5. Let eggs settle by gravity or low-speed centrifugation.

Method II (Henry, 1986)

  1. Wash in seawater.
  2. Suspend 45 s in 125 mM Na citrate, 500 mM sucrose.
  3. Wash twice in seawater.