# DNA Quantification

(Difference between revisions)
 Revision as of 00:39, 30 January 2010 (view source) (→Overview)← Previous diff Revision as of 00:43, 30 January 2010 (view source) (→Procedure)Next diff → Line 5: Line 5: == Procedure == == Procedure == - 1. Dilute the DNA sample 30X by combining the following in a cuvette + #Dilute the DNA sample 30X by combining the following in a cuvette - 87µl water + **87µl water - 3µl DNA prep + **3µl DNA prep - 2. Run the DNA quantification test on the spectrophotometerwith a dilution of 30. + #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. + #Make sure that the A260 measurement is between 0.1 and 1. - 4. If is too low then repeat the measurement using 15X dilution. + #If is too low then repeat the measurement using 15X dilution. - 84µl water + **84µl water - 6µl DNA prep + **6µl DNA prep - 5. Calculate the molar concentration of DNA using the following equation. + #Calculate the molar concentration of DNA using the following equation.
- Picomoles/µl =  DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] + *Picomoles/µl =  DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] - + - + == Notes == == Notes ==

## 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 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
1. Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30.
2. Make sure that the A260 measurement is between 0.1 and 1.
3. If is too low then repeat the measurement using 15X dilution.
• 84µl water
• 6µl DNA prep
1. Calculate the molar concentration of DNA using the following equation.</br>
• Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]