Extraction of genomic DNA

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[MATERIALS] - [INDEX]

Procedure

1. Grow two 3 L flasks of Dicty cells.
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3. Harvest cells by centrifugation at 3K for 10 min.
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5. Wash cells with Buffer 3, pellet as in step #2.
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7. Repeat wash 3 times
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9. 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 µl of Triton when cells have lysed.
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11. Incubate solution on ice for 10 minutes with occasional swirling.
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13. Pellet the nuclei at 5K for 5 minutes. The pellet should appear as a white solid with a dark center.
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15. Resuspend the pellet in 50 ml of Buffer 1 (NO TRITON), being careful not to bring along the darker material.
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17. Pellet at 5K for 5 minutes.
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19. Repeat wash if alot of dark stuff is present.
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21. 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).
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23. Gently mix the solution and add 4.0 ml 20% Sarcosyl.
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    It is very important to handle this solution carefully. Do not shake vigorously or the DNA will become sheared.
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26. Adjust the total volume of the solution to 20 ml, add it to a 250 ml flak and incubate it at 65°C for 5-10 minutes. Swirl the solution, it should become viscous.
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28. Add 20g CsCl add mix gently. It may take a while to get this in solution but be patient.
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30. 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.
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32. Centrifuge for 24 hr at 50K.
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34. Recover the pink band, extract the EthBr with salt saturated isopropanol, and dialyze the DNA against TE.
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36. 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
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• Buffer 2 (Triton Extraction Buffer)
  
  • Buffer 1 containing 0.6% Triton
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• Buffer 3 (Dicty Wash Buffer)
  
  • 0.2% NaCl
  • or use DB, or PDF
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• 20% Sarcosyl
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• 0.25 M EDTA
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