Extraction of genomic DNA

Published in Hughes and Welker 1988.

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[MATERIALS] - [INDEX]

Procedure

Note: Can use as little as one confluent plate (10⁷) or 500 ml of 10⁷ cells/ml. Volumes in parentheses are suggested volumes for 10⁸ cells. These should be adjusted if a different number of cells is used.

1. Collect cells.
2. 
   
3. Wash once in KK2.
4. 
   
5. Wash once in ice-cold 0.2% NaCl (= 0.034 M).

To decide volume to use: aim at 5-10 x10⁷ cells/ml (1 ml).

Extract nuclei:

1. Resuspend cells in ice-cold NP40-lysis buffer at 5-10 x10⁷ cells/ml (1 ml).
2. 
   
3. Vortex vigourously for 30 seconds.

Lyse nuclei:

1. Add one volume of 2% SDS (1 ml).
2. 
   
3. Incubate at 65°C for 15 minutes.
4. 
   

Purify DNA:

1. Add one volume of TE, pH 9.5 (2 ml).
2. 
   
3. Add proteinase K to 200 µg/ml.
4. 
   
5. Incubate at 45-55°C for 2h to overnight.
6. 
   
7. Cool briefly.
8. 
   
9. Extract with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1).
10. 
    
11. Repeat previous step until no more interphase is visible.
12. 
    
13. Extract with chloroform:isoamyl alcohol.
14. 
    
15. Precipitate DNA: add NaCl and ethanol, incubate at -20°C for >30 minutes.
16. 
    
17. Resuspend in an appropriate volume of TE (100 µl).
18. 
    
19. Quantify using OD₂₆₀.

[TOP] [INDEX]

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Materials

• 1x KK2
• 
  
• 0.2% NaCl
• 
  
• NP40 lysis buffer
  • 0.05M HEPES
  • 0.05 M MgAc
  • 10% sucrose
  • 2% Nonidet P40 (NP40)
  
  
• 2% SDS
• 
  
• TE, pH 9.5 (10 mM Tris, pH 9.5, 1 mM EDTA)
• 
  
• Proteinase K (10-50 mg/ml)

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