DNA Quantification

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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.

Procedure

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)]

Notes

• One mole of single base pairs weighs 660 grams.
  • One picomole of 1000bp weighs 660ng.