Quantitation of DNA is commonly performed in laboratories to assess the average concentration and measure the purity of DNA in a given sample which is an important step in DNA typing. There are several methods used to quantitate solutions of nucleic acid and using the right one is crucial in achieving optimum performance of the process.
In forensic investigations where samples can be limited, quantitation of DNA can be performed by means of fluorescent absorption and ultra-violet spectroscopy.
Here is a simple protocol for DNA Quantitation.
- Prepare a sample of diluted DNA with a final volume of 400 µL and dilution ratio of 1:100 to 1:400.
- Pre-heat the spectrometer for at least 15 minutes. When the machine is ready, set the wavelength to 260nm. This ratio of absorbance will allow the DNA to absorb the ultra-violet light once the sample is exposed inside.
- Blank the spectrophotometer by using 400 µL of the same solvent used to dilute your DNA. Fill both quartz cuvettes to determine the background constant. Remember that the quartz cuvettes are different from optical plastic cuvettes. Plastic cuvettes are only used for fast spectroscopic assays where speed is more important than high accuracy.
- Empty the cuvettes and place the diluted DNA sample in it.
- Record the OD260.
- Use this formula to determine the concentration of the original dsDNA stock solution.
OD260 x 50 µg/ mL x dilution ratio (e.g. 100 or 400) = concentration of DNA
- Clean the cuvettes with distilled water and turn off the spectrophotometer.
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