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DNA quantification
DNA quantification methods overview. Some commonly used DNA quantification methods and related resources. DNA quantification methods include UV spectrometry, fluorescent DNA-binding dyes, agarose gel electrophoresis and staining, real-time PCR etc.
DNA yield can be accurately measured or roghly estimated using different methods: UV spectrometry (absorbance), fluorescent DNA binding dye (sybr Green etc with fluoreimager), agarose gel electrophoresis (band intensity), and real-time PCR.
The UV spectrometer is used to determine the absorbance of DNA which has highest absorbance at 260nm. The A260 reading between 0.1 to 1.0 can be used to estimate the DNA quantity. A260 reading should be accompanied by A280 and A230 to determine the DNA purity since RNA, protein (at A280), and guanidine (at A230) can interfer with the DNA absorbance at 260nm. A good rule of thumb is high purity DNA should yield A260/A280 ratio of 1.7-2.0, and A260/A230 is greater than 1.5.
DNA concentration calculation
- A260 reading of 1.0 = 50 ug/ml DNA
- DNA Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml
- DNA Purity (A260/A280) = (A260 reading – A320 reading) / (A280 reading – A320 reading)
DNA agarose gel electrophoresis can also be used to estimate the quantity of the DNA by comparing its band intensity to that of a DNA standard with known quantity after ethidium bromide (EB) or SYBR Green staining of the gel.
DNA-binding dyes such as PicoGreen and Hoechst are used for DNA quantification by comparing the sample DNA to a DNA standard curve. The DNA-binding dye method is sensitive (for hoechst from 10ng/ml to 250ng/ml, for PicoGreen from 25pg/ml to 1ug/ml). When using DNA-binding dyes for DNA quantification, one has to consider the DNA dilution, length of DNA, and types of DNA (genomic, plasmid, PCR products all require different standard curve).
DNA concentration protocols:
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