Southern Hybridization

1. Prehybridization buffer: add 1 g dextran sulfate and 1 mL 10% SDS to 8.46 mL dH2O and heat at 65°C for 30 min (until everything is dissolved); then add 0.58 g NaCl and heat for an additional 15 min at 65°C, until dissolved.

2. Denatured sheared salmon sperm DNA: dilute 50 yg sheared salmon sperm DNA to 2 mg/mL in dH2O in a microfuge tube. Boil for 10 min in a beaker on a hot plate. Immediately chill in wet ice for 15 min.

3. Glass capillary tubes (Microcaps, 50 yL size, Drummond).

4. Diamond tip scriber.

5. Probe template (gel purified using Qiagen Qiaex II Gel Extraction Kit).

7. LS solution: 440 mM HEPES-NaOH, pH 7.6, 44 yM each of dTTP, dCTP, and dGTP, 110 mM Tris-HCl, pH 7.5, 11 mM MgCl2, 22 mM 2-mercaptoethanol, 300 yg/mL random oligodeoxyribonucleotide hexamers (IDT).

9. DNA Polymerase I, Large (Klenow) Fragment.

10. Kodak Biomax MR film.

11. Kodak Biomax MS intensifying screen.

12. Autoradiogram exposure cassettes.

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