Exercise 14.3 - Characterization of dna

LEVEL I

Materials

• DNA sample
  
• SSC buffer
  
• UV spectrophotometer 3 and quartz cuvettes

Procedure

1. Dissolve a small quantity of your extracted DNA in 3.0 ml of 0.1X SSC.

2. Turn on and blank a UV spectrophotometer at 220 nm (use 0.1X SSC as the blank). Determine the absorbance of your sample DNA at 230 nm.

3. Change the wavelength to 230 nm, reblank the spectrophotometer and measure the absorbance of the sample at 230 nm.

4. Increment the wavelength by 10 nm and repeat blanking and measuring the absorbance until readings are taken through 300 nm.

5. Compute the absorbance ratio 260 nm to 280 nm. Pure DNA (without protein or RNA) will have a 260:280 absorbance ratio of 1.85. RNA will have a 260:280 ratio of 2.0.

6. Plot the absorbance spectrum of your sample and indicate the 260:280 ratio, as well as the amount of protein contamination on the graph.

   Wavelength	Absorbance
   220
   230
   240
   250
   260
   270
   280
   290
   300

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