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
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
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
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
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