- 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
Note that lyophilized, highly polymerized DNA is extremely slow to go
into solution. It will require preparation at least 1 day in advance of the
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, 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 5 tubes
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
- 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