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DNA Concentration By UV Spectrophotometry Measure Absorption

Summary: DNA Concentration By UV Spectrophotometry
Measure Absorption:
1. Dilute DNA to 0.5 to 50 g/ml in TE buffer or dH2O.
Plan to use a quartz cuvette or a UV-transparent plastic (disposable) cuvette (1 cm path length).
Plan for a final volume as follows: Semi-microcuvette 500 l
Microcuvette 100 l
2. Measure absorption at 260 nm (A260). Start by zeroing instrument with TE buffer or dH2O alone.
If A260 reads less than 0.01, prepare a less dilute sample.
To estimate purity, also measure A280 (protein). Note readings for TE buffer or dH2O "blank".
Plasmid Preps:
1. Dilute 1 l of plasmid DNA into 10 l TE (dilution can be kept for digestions).
2. Add 1 l of diluted DNA to 99 l dH2O in a quartz microcuvette.
Calculate DNA Concentration:
S is the size of the DNA in kilobases. N is the number of bases A, G, C, or T.
For example, A260 = 1 for double-stranded DNA corresponds to 50 g/ml.
Pure DNA yields an A260/A280 ratio of 1.8 - 1.9. (Pure RNA yields a ratio of 1.9 - 2.0.)


Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida


Collections: Biology and Medicine