2 DNA extraction - Purity analysis and quantitation
 

 

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Equipments and reagents required

 

UV-VIS spectrophotometer

Quartz cuvettes for UV measurements

Dry wipe tissues

Micropipette

 

Double distilled water

TE buffer

 

 

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 it 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 tissue.

6.   Using a micropipette, clean the inside of the cuvette by TE buffer.

7.   Insert the cell containing 100 l of TE buffer into chamber.

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 buffer.

12. Add 5.0 I of the crude DNA sample in the cuvette. Add 95 l of TE buffer. Mix solution thoroughly.

13. Insert the cuvette into the sample compartment and close the cover tightly.

14. Read the OD value directly from the screen.

15. Calculate the DNA concentration in g/ml by multiplying the OD260 value by 1000.

     That is, if OD260 equals 0.200 then DNA concentration equals 0.200 x 50 x 20 = 200 g/ml (50 because 1 unit OD corresponds to a concentration of 50 g/ml and 20 because the DNA solution was diluted 20 X).

Assessment of the DNA purity

 

1.   Read the OD at 280 nm.

2.  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.

3.  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.