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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)]
- One mole of single base pairs weighs 660 grams.
- One picomole of 1000bp weighs 660ng.