Preparation of Nuclei

1. Defrost four frozen mouse livers in HB buffer on ice (see Note 1).

2. Drain buffer from livers. Mince livers well with surgical scissors.

3. Add minced livers and 50 mL HB buffer containing 0.1% Triton X-100 to the (pre-chilled VirTis) homogenization vessel. Homogenize the livers at half speed for 2 min.

4. Filter the homogenized livers through two layers of cheesecloth into 15-mL glass Corex centrifuge tubes (usually takes four tubes).

5. Centrifuge the nuclei in an SS-34 Sorvall rotor at 4°C, 4500g for 15 min.

6. Carefully decant supernatant and discard.

7. Resuspend the pellets in about 10 mL HB buffer per tube (see Note 2).

8. Homogenize the resuspended pellets using a (prechilled) Dounce homogenizer with a loose-fitting pestle, using about four strokes of the pestle.

9. Transfer the homogenized nuclei to clean prechilled Corex tubes and centrifuge again at 2300g for 10 min.

10. Again discard the supernatant, resuspend pellets in HB Buffer, and Dounce homogenize as above.

11. Centrifuge again as before. Pellets should appear to be a creamy off-white to light tan color. The wash with HB buffer can be repeated once more if necessary.

12. After the final wash and homogenization, resuspend the nuclei very gently in 0.1 M TE to a total volume of 20 mL; do not Dounce homogenize (see Note 3).

13. To measure the approximate DNA concentration of the nuclear suspension:

a. Remove 10 ^L of the suspension to a new tube, immediately after dispersal by gentle agitation, using a cut-off pipet tip.

b. Add 990 ^L of 0.1 NNaOH to the 10 ^L of nuclei, heat to 95°C for 5 min, mix well, and measure the A260 value.

c. Calculate the approximate concentration (mg DNA/mL) of the undiluted suspension using an A260 value of 28 for denatured DNA, and assuming that about 50% of the A260 arises from RNA.

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