1. Collect embryos for 24h. Optional: Let them age for up to 12h at room
temperature.
2. Decorionate embryos for 2 minutes in 100% bleach fluid (2% sodium
perchloride).
3. Wash embryos with 1 x PBS.
4. Wash embryos with 1 x PBT.
5. Transfer embryos into a bottle containing 1 volume of PBS and 1 volume of n-
Heptane. Use 20 ml of PBS per 1 ml of embryos. Mix by briefly shaking the
bottle.
6. Remove PBS (lower phase). Leave the interphase intact.
7. Add 1 volume of methanol and shake vigorously by hand for 1 minute.
8. Remove n-heptane and interphase.
9. Transfer embryos into the Falcon tube (15ml or 50ml depending on the
sample volume) and wash twice with 1 volume of methanol.
10. Remove methanol.
11. Wash embryos with 1 volume of PBS.
12. Add 1 volume of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM
NaCl, 0.5% SDS, 5 mg/ml Proteinase K, 100 mg/ml RNAse A).
Lyse for 2-3 hours at 55°C. Gently mix by inverting the tube every 15 minutes.
13. Centrifuge at 4000g for 15 minutes. Transfer supernatant to a new Falcon
tube. Optional: Remove 200 μl for quality analysis.
14. Add 1 volume of Phenol:Chloroform:Isoamyl alcohol. Incubate on
a rotating wheel for 1 hour at 4°C.
15. Centrifuge at 4000g for 10 minutes. Transfer aqueous (upper) phase to
a new Falcon tube.
16. Repeat steps 14–15.
17. Add 1 volume of Chloroform:Isoamyl alcohol. Incubate on a rotating wheel for
1 hour at 4°C.
18. Centrifuge at 4000g for 10 minutes. Transfer aqueous (upper) phase to
a new Falcon tube.
19. Add 0.05 volume of 3.0 M KAc. Mix by gently inverting the tube.
20. Add 0.7 volume of isopropanol. Incubate on a rotating wheel for 30 minutes at
4°C.
21. Centrifuge at 6000g for 15 minutes. Remove supernatant.
22. Wash the pellet twice with 1 volume of 70% ethanol.
23. Air-dry the pellet for 10 minutes at room temperature.
24. Dissolve the pellet in 1 x TE prewarmed to 55°C. Store DNA at 4°C.