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Quantitation of DNA and RNA

Adapted from "General Procedures," Appendix 3, in Phage Display, by Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001.

INTRODUCTION

There are several ways to quantitate solutions of nucleic acids. If the solution is pure, one can use a spectrophotometer to measure the amount of ultraviolet radiation absorbed by the bases. DNA can also be quantified by measuring the UV-induced emission of fluorescence from intercalated ethidium bromide. This method is useful if there is not enough DNA to quantify with a spectrophotometer, or if the DNA solution is contaminated. Strategies for accurately quantifying nucleic acids using these approaches are discussed here.

QUANTITATION WITH A SPECTROPHOTOMETER

Use H₂O or 1X TE as a solvent to suspend the nucleic acids, and place each sample in a quartz cuvette. Zero the spectrophotometer with a sample of solvent. For more accurate readings of the nucleic acid sample of interest, dilute the sample to give readings between 0.1 and 1.0.

For a 1-cm pathlength, the optical density at 260 nm (OD₂₆₀) equals 1.0 for the following solutions:

• a 50 µg/mL solution of dsDNA
  
  
• a 33 µg/mL solution of ssDNA
  
  
• a 20-30 µg/mL solution of oligonucleotide
  
  
• a 40 µg/mL solution of RNA
  
  

Contamination of nucleic acid solutions makes spectrophotometric quantitation inaccurate. Calculate the OD₂₆₀/OD₂₈₀ ratio for an indication of nucleic acid purity. Pure DNA has an OD₂₆₀/OD₂₈₀ ratio of ~1.8; pure RNA has an OD₂₆₀/OD₂₈₀ ratio of ~2.0. Low ratios could be caused by protein or phenol contamination.

Example of Calculation

A sample of dsDNA was diluted 50X. The diluted sample gave a reading of 0.65 on a spectrophotometer at OD₂₆₀. To determine the concentration of DNA in the original sample, perform the following calculation:

dsDNA concentration = 50 µg/mL x OD₂₆₀ x dilution factor

dsDNA concentration = 50 µg/mL x 0.65 x 50

dsDNA concentration = 1.63 mg/mL

QUANTITATION BY ETHIDIUM BROMIDE FLUORESCENCE EMISSION

Run DNA samples (include several amounts ranging from 25 to 200 ng) on a 0.8% agarose minigel containing 0.5 µg/mL ethidium bromide. Run the samples next to DNA standards of known concentration or use molecular mass markers (DNA Mass Ladders, Invitrogen, 10068-013 or 10496-016). To maintain constant background staining of the gel, include 0.5 µg/mL ethidium bromide in the running buffer.

Use a UV light to photograph the gel. Compare fluorescence intensities and estimate DNA concentrations.

To quantitate mixtures of DNA fragments or DNA of multiple sizes, spot samples on a 1% agarose slab gel containing 0.5 µg/mL ethidium bromide. Also spot several DNA standards of known concentration. Let the gel stand for a few hours at room temperature to allow small contaminating molecules to diffuse away. Use a UV light to photograph the gel. Compare fluorescence intensities and estimate nucleic acid concentrations.