MNase can cut DNA non-randomly. therefore, to determine how this naturally non-random pattern is affected by nuclear proteins such as histones, it is important to compare the MNase cutting pattern of chromatin with the MNase cutting pattern of free genomic DNA. Source Compact Laboratory Guide to Molecular Biology, Fifth Edition

Operation method

Nuclease digestion of purified genomic DNA by Micrococcus spp.

Materials and Instruments

0.5 to 1 mg purified genomic DNA Micrococcus nuclease (MNase)
Nuclear buffer C 0.5 mol L CaCl2 0.25 mol L EGTA Chloroform
Heater set at 68°C Ice

Move

1. Take 100 ug of genomic DNA and add it to room temperature Nuclear Buffer C to a final volume of 300 ug. Prepare 4 to 5 tubes of this solution.

IMPORTANT: For each solution, do not proceed to the next tube until the reaction has reached Step 4. 2.

2. Add _CaCl2 to a final concentration of 3 mmol/L. 3.

3. add MNase to 0.5, 1, 2, 3 U/ml. mix vigorously to distribute the enzyme evenly (the solution may be somewhat viscous) and react for 3 min.

4. Transfer the solution to a tube containing 50 ul of 0.25 mol/L EGTA preheated to 68°C. Incubate at 68°C for 10 min.

5. Transfer the tube to ice. 6.

6. 200 ng of sample is loaded onto a 1.2% mini agarose gel and the extent of digestion is detected by electrophoresis. 7.

7. For samples of interest, add an equal volume of chloroform and extract once. 8.

8. DNA is precipitated, quantified and characterized.

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