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Method: Preparation of Sonicated Human DNA

June 7, 1990

Srini Ramachandra


Purpose:

Time required:

Special reagents required:

Special equipment required:

Procedure:

  1. Dissolve 500 mg human placental DNA in 100 ml TE overnight at room temperature.

    Use a sterile beaker and keep covered.

  2. Transfer dissolved DNA in 8 ml aliquots to 15 ml screw-cap tubes and set up the sonicator controls as follows: Output control = '6'; Duty cycle = '60% '; Hold = 'continuous'.
  3. Wash the micro-tip with 70% EtOH and dH2O. Wipe dry with Kimwipes. Place the micro-tip immersed halfway in the DNA solution. Wear ear muffs.
  4. Sonicate the DNA in 1 minute pulses for 7 to 10 minutes with a 1 minute cooling interval between each pulse. (i.e. sonicate for 1 minute, cool for 1 minute). During this cooling interval place the tubes in an ice bucket. Wash the micro-tip with 70% EtOH and ddH2O before and after each use.
  5. Combine the samples and run 1 l of sonicated DNA on a 0.8% TA-agarose gel at 50V for 1 hour with BRL 1kb ladder as a marker.
  6. The bulk of the sonicated DNA should be around 500 bp in size. If not, continue sonication and testing samples on the gel until you have the right size fragments.
  7. Take a spectrophotometer reading of the sonicated DNA and adjust the concentration to 2.5 mg/ml (see sample calculation below). Take a small aliquot of the DNA and dilute to produce an OD260 reading to between 0.1 and 0.3.

    Example:

  8. Adjust the DNA concentration to 2.5 mg/ml with TE. Store in 5 ml aliquots at -20 degrees C.

References: none