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Chemical Devitellinization Protocol

(Paul Chang '97)
  1. Prepare 20 ml of 0.1% trypsin in 18 mM CaCl2 HTR, pH 8.2
  2. Aliquot into 20, 1 ml eppendorf tubes; store aliquots in -20c freezer until use
  3. Immediately before use, make sure solution is fully thawed, and at room temperature; add 10 mM DTT to each aliquot (0.0015 g per 1 ml)
  4. Prepare 3 agarose covered petri dishes
  5. Fill one with devitellinization solution; fill other two with 18 mM CaCl2 HTR penicllin/streptomycin/gentamycin solution for washing
  6. Flame 4 Pasteur pipettes to blunt sharp edges
  7. Place embryos in devitellinization solution
  8. Watch embryos carefully; when vitelline envelope begins to lift off embryo and break open, transfer embryo into wash solution
  9. Each embryo will "break" at different times, treat each as an individual
  10. As soon as all embryos are in wash, transfer to new wash dish
  11. Wait 5 minutes between washes

Manual Devitellinization Protocol

(Bob Goldstein '98)

Method #1: Using Insect Pins

Fixed embryos can be manually devitellinized one at a time with insect pins or more quickly in bulk with a narrow-bore pipet. The narrow-bore pipet method will damage some embryos (<10% of the embryos when done well). Damaged embryos are discarded and the remainder are used.

Method #2: Using Pasteur Pipettes

  1. Prepare a narrow-bore pipet by pulling the ends of 5-10 glass pasteur pipets over a flame:
  2. Using watchmakers' forceps, cut the end of each pipette to a diameter roughly just smaller than the diameter of vitelline envelopes: Under the dissecting scope, place pipette next to some embryos (for size comparison). Start by cutting at a diameter a bit too small, and then work the way up the pipette a little at a time, breaking off pieces until the opening is the right diameter. Jagged edges are okay.
  3. After wash, fixed embryos can then be devitellinized by pulling and pushing them into and out of the narrow bore pipet, one batch at a time. Make sure the dish has plenty of liquid or air will make loads of bubbles. Embryos will get stuck and/or damaged in pipets with bores that are too narrow, and will not be devitellinized in pipettes with bores that are too large. Rinse and re-use the best narrow bore pipet of the 5-10 made and discard the rest.