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(Source: M.-A. Akimenko from Zebrafish Book 5th Edition)

Modified from Blin and Stanford Nucl. Acids Res. 3:2303-2308 (1976)

1. Grind frozen adult zebrafish to fine powder in liquid nitrogen.

2. Suspend in 10 ml of digestion buffer: 10 mM Tris-HCl pH 8.0. 1% SDS, 5 mM EDTA, 100 μg/ml of proteinase K.

3. Digest at 37°C for 5 hours with shaking.

4. Extract once with an equal volume of phenol followed by two extractions with an equal volume of phenol:chloroform:isoamyl alcohol (50:48:2).

5. Adjust the NaCl concentration of the aqueous phase to 0.3 M NaCl and precipitate with 2.5 volumes of ethanol.

6. Mix by inversion and centrifuge immediately for 10 minutes at 3000 x g.

7. Rinse pellet with 70% ethanol and dry.

8. Resuspend the pellet in 1 ml of 10 mM Tris-HCl pH 8.0, 1 mM EDTA.

9. Add NaCl to a final concentration of 100 mM and incubate with 100 μg/ml of deoxyribonuclease-free ribonuclease A at 37°C for 3 hours.

10. Add SDS to a final concentration of 0.2% and extract the solution twice with an equal volume of phenol:chloroform:isoamyl alcohol (50:48:2) followed by precipitation with ethanol as in step 5.

11. Resuspend the DNA pellet in 1 ml of 10 mM Tris-HCl pH 8.0, 1 mM EDTA.