Chromatin extraction

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use pellet from cytoplasm extraction

  1. Chromatin lysis buffer
    • 10mM Hepes or Tris (pH7.5)
    • 10mM KCl
    • 10% glycerol
    • 1mM NaPPi
    • 1ug/mL pepstatin
    • 1ug/mL aprotinin
    • 1ug/mL leupeptin
    • 1mM NaVO4
    • 1mM NaF
    • 1mM PMSF
    • water to 50 mL

Resuspend pellet from cytoplasm extraction in 2mL of chromatin extraction buffer. Add 50µg/mL Digitonin (50mg/mL stock—add 3µL). Keep on ice for 1 hour. Centrifuge at 2000rpm for 5 minutes. Wash pellet in chromatin buffer twice. Resuspend pellet in 200µL of Chromatin Buffer (may have to increase volume for larger pellet. Increase volume of DNAse accordingly). Add 3µL of DNAse and incubate for 2hours at 4∞C with mixing.

  1. 2X high salt buffer
    • 300mMNaCl
    • 2% Triton-X100
    • 100mM Hepes or Tris (pH7.5)
    • 20% glycerol
    • 1mM NaPPi
    • 1ug/mL pepstatin
    • 1ug/mL aprotinin
    • 1ug/mL leupeptin
    • 1mM NaVO4
    • 1mM NaF
    • 1mM PMSF
    • water to 50 mL

Add same volume of 2x High Salt Buffer. Incubate for 1 hour on ice. Centrifuge for 15 minutes at 14,000rpm at 4∞C. Keep Supernatant.

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