Extraction of genomic DNA (CsCl)

Extraction of genomic DNA

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Procedure

  1. Grow two 3 L flasks of Dicty cells.

  2. Harvest cells by centrifugation at 3K for 10 min.

  3. Wash cells with Buffer 3, pellet as in step #2.

  4. Repeat wash 3 times

  5. Following the last spin wipe all buffer from the tubes and resuspend the cells in 100 ml of Buffer 2 (Triton Buffer). Add an additional 700 l of Triton when cells have lysed.

  6. Incubate solution on ice for 10 minutes with occasional swirling.

  7. Pellet the nuclei at 5K for 5 minutes. The pellet should appear as a white solid with a dark center.

  8. Resuspend the pellet in 50 ml of Buffer 1 (NO TRITON), being careful not to bring along the darker material.

  9. Pellet at 5K for 5 minutes.

  10. Repeat wash if alot of dark stuff is present.

  11. Resuspend the pellet in 6.0 ml water then add 8.0 ml 0.25 M EDTA (the total volume should be about 16 ml).

  12. Gently mix the solution and add 4.0 ml 20% Sarcosyl.

  13. It is very important to handle this solution carefully. Do not shake vigorously or the DNA will become sheared.

  14. Adjust the total volume of the solution to 20 ml, add it to a 250 ml flak and incubate it at 65C for 5-10 minutes. Swirl the solution, it should become viscous.

  15. Add 20g CsCl add mix gently. It may take a while to get this in solution but be patient.

  16. Once the CsCl has dissolved, add 2.0 ml of Ethidium Bromide (10 mg/ml) and load into two ultracentrifuge quick seal tubes. The solution should be viscous, so be careful not to glob it all over.

  17. Centrifuge for 24 hr at 50K.

  18. Recover the pink band, extract the EthBr with salt saturated isopropanol, and dialyze the DNA against TE.

  19. To concentrate, ethanol precipitate.

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Materials

  • Buffer 1 (Extraction Buffer)
    • 50 mM Tris; pH 8.0
    • 10 mM NaCl
    • 3 mM MgCl2
    • 3 mM CaCl2
    • 0.5 M Sorbitol

  • Buffer 2 (Triton Extraction Buffer)
    • Buffer 1 containing 0.6% Triton

  • Buffer 3 (Dicty Wash Buffer)
    • 0.2% NaCl
    • or use DB, or PDF

  • 20% Sarcosyl

  • 0.25 M EDTA

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