© Michael Court
Jan 2003
RNA and DNA concentration by UV absorbance
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Turn on UV spectrophotometer and let warm it up for at least 15-20 minutes.
a. Use fixed wavelength program and set wavelength to 260 nm
Dilute sample with TE pH 8.0 buffer in a microcentrifuge tube
a. For RNA add 2 uL sample to 48 uL TE buffer (1/25 dilution)
b. For DNA add 3 uL sample to 57 uL TE buffer (1/20 dilution)
Blank (zero) spectrophotometer with 50 ul cuvette containing TE buffer
a. Wash cuvette with distilled water before and between samples
b. Use cuvette holder closest to you
c. Ensure that cuvette is oriented such that the light beam passes through the window in
cuvette side left to right
d. Alternative – can also read absorbance of blank and subtract this reading from all
other readings
Add sample to cuvette and read absorbance
a. Absorbance equals the nucleic acid concentration in ug / uL (mg/mL)
i. For example a reading of 0.5 means 0.5 ug / uL
b. Dilute sample further if reading is greater than 2.0
i. Don’t forget to adjust concentration for dilution
ii. Multiply OD by dilution factor
To assess DNA/RNA contamination with protein or purification chemicals a reading at
280 nm can also be obtained and used to calculate A260/A280 ratio
a. Best to get blank (zero) value at both 260 and 280 nm and subtract from readings
b. Ratios should be between 1.5 and 2.0 (ideally 1.6 to 1.8)
i. Low ratios indicate significant presence of protein or other contaminant
ii. High ratios can occur if inappropriate diluent is used (unbuffered water)