Exercise 14.4 - DNA - Dische Diphenylamine Determination

LEVEL I

Material

Procedure

  1. Weigh out 15.0 mg of commercial lyophilized DNA and prepare a stock solution of 3.0 mg/ml by dissolving the DNA in 5.0 ml of SSC. This material will be used to prepare a standard curve for the diphenylamine reaction.
    Note that lyophilized, highly polymerized DNA is extremely slow to go into solution. It will require preparation at least one day in advance of lab with constant shaking.

  2. Prepare a series of known standard solutions by serially diluting the stock solution of DNA. Set up a series of test tubes containing 2.0 ml of SSC each. Pipette 2.0 ml of stock solution into tube #1, mix and pipette 2.0 ml of the resulting mixture into tube #2 and so on. This will yield a series of tubes containing 1.5, 0.75 and 0.375 mg/ml of DNA. Your original stock solution is 3.0 mg/ml and SSC should be used for the blank.

  3. Remove and discard 2.0 ml of the final dilution. To each of the five tubes containing in step 2 (each should contain only 2.0 ml), add exactly 4.0 ml of Dische diphenylamine reagent and mix well.

    This Reagent contains glacial acetic acid. It is caustic and should be handled with care.

  4. Place a marble on the top of each test tube (it should not fall into the test tube, as it will act as a reflux to prevent evaporation, while allowing for pressure changes). Place the tubes in a boiling water bath for 10 minutes, remove from the bath and immediately immerse in an ice bath to cool.

  5. Turn on a spectrophotometer and adjust the wavelength to 650 nm. Use the tube containing no DNA from step 2 to blank the instrument and measure the absorbance of each of your standards.
    Plot the absorbance against DNA concentration, perform a linear regression of the data and compute the extinction coefficient.

  6. Dissolve your extracted or sample DNA in 10 ml of SSC. Make serial dilutions of 1/10, 1/100 and 1/1000 with SSC. Measure the absorbance of your extracted or sample DNA dilutions and calculate the concentration of DNA in the sample. Use the dilution which gives an absorbance in the 0.1 to 1.5 range.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu