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 Spectrophotometry
• Used in the measurement
of Nucleic acid quality
• Test for purity
• DNA absorbs light at 260
nm
• Proteins absorb light at
280 nm
 Spectrophotometry
• Pure preparation of DNA
has OD260/OD280
• Pure DNA extract has a ratio
of 1.8-2.0
• <1.8 = contaminated with
proteins
• >2.0 = contaminated with
phenol
 Spectrophotometry
• DNA concentration at 1cm
absorbance at 260 nm =
50ug/ml
• RNA concentration at 1cm
absorbance at 260 nm =
40ug/ml
• Absorptivity constants
DNA purity/Concentration test
In a cuvette with 1cm path length a solution of
double-stranded DNA gives a reading of 0.457 at
260 nm and 0.246 at 280 nm. Assuming that 50
ug/ml DNA solution gives an absorbance of 1.000 at
260 nm. What is the concentration of the DNA
sample

Is the DNA sample pure?

DNA purity/Concentration test
In a cuvette with 1cm path length a solution of
double-stranded DNA gives a reading of 0.457 at
260 nm and 0.246 at 280 nm. Assuming that 50
ug/ml DNA solution gives an absorbance of 1.000 at
260 nm. What is the concentration of the DNA
sample?

Is the DNA sample pure?

DNA purity/Concentration test
In a cuvette with 1cm path length a solution of
double-stranded DNA gives a reading of 0.457 at
260 nm and 0.246 at 280 nm. Assuming that 50
ug/ml DNA solution gives an absorbance of 1.000 at
260 nm. What is the concentration of the DNA
sample?

Is the DNA sample pure?

Concentration
In a cuvette with 1cm path length a solution of double-stranded DNA
gives a reading of 0.457 at 260 nm and 0.246 at 280 nm. Assuming that
50 ug/ml DNA solution gives an absorbance of 1.000 at 260 nm. What
is the concentration of the DNA sample?

Concentration = 0.457 x 50 ug/ml

Concentration = 22.85 ug/ml
Concentration
In a cuvette with 1cm path length a solution of double-stranded DNA
gives a reading of 0.457 at 260 nm and 0.246 at 280 nm. Assuming that
50 ug/ml DNA solution gives an absorbance of 1.000 at 260 nm.

Is the DNA sample pure?

OD260 0.457
OD280 0.246 1.857
DNA purity: 1.8-2.0 DNA is pure