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Chemical Devitellinization Protocol
(Paul Chang '97)
- Prepare 20 ml of 0.1% trypsin in 18 mM CaCl2 HTR, pH 8.2
- Aliquot into 20, 1 ml eppendorf tubes; store aliquots in -20c freezer until use
- 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)
- Prepare 3 agarose covered petri dishes
- Fill one with devitellinization solution; fill other two with 18 mM CaCl2 HTR penicllin/streptomycin/gentamycin solution for washing
- Flame 4 Pasteur pipettes to blunt sharp edges
- Place embryos in devitellinization solution
- Watch embryos carefully; when vitelline envelope begins to lift off embryo and break open, transfer embryo into wash solution
- Each embryo will "break" at different times, treat each as an individual
- As soon as all embryos are in wash, transfer to new wash dish
- 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
- Prepare a narrow-bore pipet by pulling the ends of 5-10 glass pasteur pipets over a flame:
- 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.
- 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.