PreparationofSonicatedHumanDNA

Purpose:

• To break up high molecular weight human placental DNA into fragment sizes of 500 bp or less which can be used as competitor DNA in Southern and in situ hybridizations. This method can also be used for sonicating salmon sperm DNA.

Time required:

• 2 hours for the sonication

Special reagents required:

• human placental DNA (Sigma, Cat.# D 4642)

Special equipment required:

• A sonicator set up with a micro-tip. This method is designed for use with the sonicator on the 8th floor of McDonnell Science Building (Genetics Dept.). If you are using another sonicator please check the relevant manuals, especially about the output and duty cycle limits on the micro-tip.

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 祃 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:

   
   

   
   

• The OD260 of a sample is 0.28 at 1:300 dilution. Then:

OD260 of undiluted solution = 0.28 x 300 = 84.0

Since an OD260 reading of 1.0 = 50 礸/ml of DNA, the concentration of the undiluted solution is 50 x 84 = 4200 礸/ml, or 4.2 mg/ml.

If the present volume is 100 ml (420 mg), add enough TE to adjust the final concentration to 2.5 mg/ml. (In this case, add 68 ml TE to bring the final volume to 168 ml.)




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