Extraction of genomic DNA

Cytoskeleton isolation

Published in Reines and Clarke 1985.

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[MATERIALS] - [INDEX]

Procedure

  1. Harvest 2 x 107 cells.

  2. Add 1 ml Lysis buffer.

  3. Sit on ice for 2 min.

  4. Spin in microfuge at 15k x 5 min.

  5. Wash pellet with 0.5 ml Lysis buffer and then 0.5 ml Wash buffer.


The pellet contains actomyosin cytoskeletons. To release myosin from the pellet:

  1. Resuspend pellet in 50 ┬Ál Release buffer.

  2. Incubate on ice for 5 min.

  3. Spin in microfuge 15K x 5 min.

  4. The supernatant contains myosin released from cytoskeletons. For Western blot, 1/5 of the supernatant is plenty.

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Materials

  • Wash buffer
    • 100 mM Pipes pH 6.8
    • 2.5 mM EGTA
    • 1 mM MgCl2
    • 10 mM TAME
    • 20 mM Benzamine


  • Lysis buffer
  • Wash buffer + 0.5% Triton


  • Release Buffer
  • Wash buffer + 200 mM KCl + 2 mM ATP

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