Overview
This protocol uses a spectrophotometer to quantify the amount (μg/mL) of DNA and then uses a simple equation to convert this mass concentration into a molar concentration. The molar concentration is much more useful for most enzymatic processes.
Example: digesting 500ng of a 2KB plasmid is twice as much "enzymatic work" as digesting 500ng of a 4KB plasmid with the same multiple cloning site.
1. Dilute the DNA sample 30X by combining the following in a cuvette:
87µl water
3µl DNA prep
2. Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30.
3. Make sure that the A260 measurement is between 0.1 and 1.
4. If is too low then repeat the measurement using 15X dilution.
84µl water
6µl DNA prep
5. Calculate the molar concentration of DNA using the following equation:
Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]