Exercise 14.4 - DNA - Dische Diphenylamine Determination
- Lyophilized DNA standard
- Sample DNA
- Dische diphenylamine reagent
- 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.
- 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.
- 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
This Reagent contains glacial acetic acid. It is caustic and
should be handled with care.
- 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.
- 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.
- 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 -- firstname.lastname@example.org