Exercise 14.2 - Purification of DNA

LEVEL I

Materials

• Spooled DNA from Exercise 14.1
  
• SSC buffer
  
• Pancreatic Ribonuclease A (100 µg/ml)
  
• Pronase
  
• Sodium lauryl sulfate (SLS)
  
• Sodium perchlorate
  
• Chloroform:isoamyl alcohol (24:1)
  
• 95% and 70% (v/v) ethanol
  
• Refrigerated centrifuge, rotor and tubes

Procedure 1

1. Decant off the alcohol, and dissolve the extracted DNA in 30 ml. of diluted SSC buffer (0.1 X SSC) in a 125 ml. erlenmeyer flask. This will require some time, as polymerized DNA dissolves slowly. Gentle swirling of the material will help.

2. Add pancreatic ribonuclease A to a final concentration of 100 micrograms/ml and agitate slowly at 37° C for 1 hour.

3. Add pronase to a final concentration of 50 micrograms/ml and again agitate slowly at 37° C for another hour.

4. Add sodium lauryl sulfate (SLS) 2 to make a 1% concentration (w/v) and sodium perchlorate to a final concentration of 1 M. Agitate for 30 minutes at room temperature.

5. Extract the solution with chloroform:isoamyl alcohol (24:1 v/v) by adding an equal volume and shaking virogously for at least 15 minutes.

6. Place the solution into appropriate centrifuge tubes and centrifuge for 5 minutes at 800 xg and 4° C.

7. Remove the upper aqueous phase, add 2.3 volumes of 95% cold ethanol and respool the DNA from this solution onto a glass rod.

8. Wash the spooled DNA twice with cold 70% ethanol and store for future analysis.

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