Mouse Liver Nuclei Prep

1. HB buffer: 0.34 M sucrose (Ultra Pure, Gibco BRL), 15 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine (14).

2. 20% (v/v) Triton X-100 Stock made by dilution of Triton X-100 with water.

3. Cheesecloth.

4. Surgical scissors.

5. Frozen mouse livers.

6. VirTis mechanical homogenizer.

7. Dounce homogenizer with loose-fitting pestle B (Bellco).

8. 2X Pro-K Stop Solution: 20 mM EDTA, pH 8.0, 0.2% sodium dodecyl sulfate (SDS), 0.5 mg Pro-K/mL.

9. 15-mL Corex glass centrifuge tubes.

10. Sorvall SS-34 rotor.

11. Adaptors for use of 15-mL tubes in the SS-34 rotor.

12. Micrococcal nuclease (Worthington): dissolved in water at 10 U/mL; small portions are stored frozen.

13. 25:24:1 Phenol/chloroform/isoamyl alcohol.

14. 24:1 Chloroform/isoamyl alcohol.

15. Ethanol (200 proof).

2.2. DNA Marker Labeling

1. 5 ^g Double-stranded DNA marker with suitable ends for labeling.

2. 10X DNA Polymerase I, Large (Klenow) Fragment buffer, 10 mg bovine serum albumin (BSA)/mL.

3. 5 U DNA Polymerase I, Large (Klenow) Fragment.

5. DEAE Sephadex A-50.

6. 1000-^L Blue pipet tip plugged with glass wool.

7. 0.2 TE: 0.2 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

8. 1.0 TE: 1.0 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

2.3. PCR Amplification of Mouse Genomic DNA

1. 0.4 mg/mL PCR oligonucleotide primers, forward and reverse (IDT).

4. 10X Taq Polymerase reaction buffer, MgCl2-free.

6. Approx 30 ^g/mL Mouse genomic DNA.

2.4. Blotting

1. Gel preparation: 200 mL of 0.25 N HCl; 200 mL of 0.4 N Na0H/0.6 M NaCl; 200 mL of 1.5 M NaCl/0.5 M Tris-HCl pH 8.0.

2. Blotting by capillary transfer.

b. Glass dish, cellulose sponge.

c. GeneScreen Plus charged nylon hybridization transfer membrane (Perkin-Elmer).

d. Whatman 3 MM filter paper, cut and stacked paper towels.

e. Glass or Plexiglas plate of size slightly larger than that of the nylon membrane.

f. 500 g weight arranged to provide even pressure over the membrane.

g. Plastic wrap.

h. Small level.

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