and reagents required
Quartz cuvettes for UV measurements
Dry wipe tissues
Double distilled water
Determination of DNA quantity
(Caution: Procedure may vary between different makes of
Spectrophotometers, but general protocol remains the same)
1. Switch on the spectrophotometer and allow
to warm up.
2. Turn on the mode to UV.
4. Set the wavelength to 280 nm.
5. Wash the cuvette with distilled H2O. Dry with
6. Using a micropipette, clean the inside of the cuvette by TE
7. Insert the cell containing 100 µl of TE
8. Set the reading to zero.
9. Set wavelength to 260
nm and then set the reading to zero.
10. Remove the cuvette from its compartment and discard the TE
12. Add 5.0 µI of the
DNA sample in the cuvette. Add 95 µl of TE
Mix solution thoroughly.
13. Insert the cuvette into the sample compartment and close the
14. Read the OD value directly from the screen.
Calculate the DNA
the OD260 value by 1000.
That is, if
OD260 equals 0.200
then DNA concentration equals 0.200 x 50 x 20 = 200 µg/ml (50
because 1 unit OD corresponds to a concentration of 50 µg/ml and 20
because the DNA solution was diluted 20 X).
Assessment of the DNA purity
Read the OD at 280 nm.
Compute for OD260/OD280. A ratio value of 1.8 suggests a highly
pure preparation of DNA. Ratio values much less than that implies
significant presence of contaminants (generally proteins) such that
accurate quantitation of nucleic acids is not guaranteed.
Read the OD values for the other samples at 260 and 280 nm by
repeating the steps described above. Make sure to wash the cuvette
thoroughly with TE between DNA samples.