Exercise 14.3 - Characterization of dna
- DNA sample
- SSC buffer
- UV spectrophotometer 3 and quartz cuvettes
- Dissolve a small quantity of your extracted DNA in 3.0 ml of 0.1X SSC.
- 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.
- Change the wavelength to 230 nm, reblank the spectrophotometer and measure the absorbance of the sample at 230 nm.
- Increment the wavelength by 10 nm and repeat blanking and measuring the absorbance until readings are taken through 300 nm.
- 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.
- 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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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org