DNA Extraction from Precipitated Chromatin (3 h)

70. Add 500 µl of phenol:chloroform:isoamyl alcohol (25:24:1, v:v:v) to the bound (Step 69) and unbound (Step 58) fractions.
The DNA extractions from the immunoprecipitated chromatin fractions might be slightly enhanced by including a proteinase K (PK) digestion step just before phenol:chloroform:isoamyl alcohol extraction. For PK digestion, add PK to each sample (to a final concentration of 100 µg/ml), and incubate the samples for 1 hour at 50°C.

71. Vortex the samples for 30 seconds.

72. Centrifuge the samples at 13,000 rpm (~15,000g) for 15 minutes in a microcentrifuge.

73. Carefully transfer the upper (aqueous) phase to another 2-ml microcentrifuge tube.

74. Add NaCl to a final concentration of 250 mM.

75. Add 10-20 µg of glycogen, and mix.
Because the DNA concentration in the bound fraction is usually low, we recommend the use of glycogen as a coprecipitator.

76. Add 1 volume of isopropanol, and mix.

77. Keep the samples for at least 2 hours at -80°C.

78. Centrifuge the samples at 13,000 rpm in a microcentrifuge for 30 minutes. Carefully discard the supernatant.

79. Rinse each pellet with 1 ml of 70% (v/v) ethanol.

80. Centrifuge the samples at 13,000 rpm for 5 minutes in a microcentrifuge. Carefully discard the supernatant.

81. Dry the pellets for 5-10 minutes at room temperature, and resuspend each pellet in 10-50 µl of 1X TE buffer. The DNA samples can be stored at 4°C.

Assessment of Precipitated Chromatin

82. Measure the OD₂₆₀ of each sample (from Step 81) in order to calculate how much DNA to use as a template in the subsequent PCR amplification (see PCR-Based Analysis of Immunoprecipitated Chromatin).
The ratio of the DNA in the bound fraction versus the total starting material (corresponding to the bound and unbound fractions together; this value was obtained at Step 37) indicates the efficiency of the ChIP assay, as it represents the percentage of immunoprecipitated chromatin. In a standard analysis of histone modifications, no more than 15% of the input native chromatin should be precipitated. However, this depends on the nature and the abundance of the histone modifications, and on the characteristics and concentrations of the antibodies used (see Discussion).

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