This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

CHAPTER 9 - MOLECULAR METHODS

Preparation of Genomic DNA for Southern Analysis

(Source: M.-A. Akimenko)

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 hr with shaking.

4. Extract once with an equal volume of phenol followed by two extractions with an equal volume of phenol:chloroform:iso-amyl 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 min 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 hr.

10. Add SDS to a final concentration of 0.2% and extract the solution twice with an equal volume of phenol:chloroform:iso-amyl 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.

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