MNase Digest of Mouse Liver Nuclei and DNA Purification

First, perform a small-scale test MNase digest of the nuclei:

1. Add 20 ^L 2X Pro-K Stop Solution to each of five microfuge tubes (labeled time points 0, 1, 2, 3, and 5 min).

2. Add 0.10 M CaCl2 to a 100-^L aliquot of the nuclei for a final concentration of 2 mM (see Note 4).

4. Withdraw a 20-^L aliquot at the 0-min time point, and add it to the tube of Pro-K marked 0.

5. Add 4 U of MNase to the remaining 80 ^L of nuclei and mix well by pipeting up and down several times. Incubate at 37°C and withdraw 20-^L aliquots at 1, 2, 3, and 5 min, plunging each aliquot immediately into the corresponding tube of 2X Pro-K.

6. Incubate in the Pro-K for about 30 min to 1 h in a heating block at 50°C.

7. Add 1/10 vol of 1 M Tris-HCl, pH 8.0, to each tube, and extract with equal volumes of 25:24:1 phenol/chloroform/isoamyl alcohol, and then with 24:1 chloroform/ isoamyl alcohol.

8. Adjust to 0.2 MNaCl (using a 5 M stock), and ethanol precipitate with 2 vol of etha-nol. Microfuge to collect the precipitated DNA.

9. Dissolve each DNA pellet in 100 ^L 1X gel loading buffer (plus RNase) and incubate for about 30 min at room temperature.

10. Run 10 ^L of each time point, along with appropriate DNA size markers, on a 1.5% agarose minigel at 100 V for about 1 h to check extent of digestion of each time point.

Once the MNase digestion conditions have been optimized using small-scale digests, perform the large-scale MNase digests of the nuclei using those conditions scaled up proportionately. The large-scale digests should be incubated in the Pro-K Stop Solution overnight at 37°C.

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