# DNA Quantification

(Difference between revisions)
 Revision as of 00:43, 30 January 2010 (view source) (→Procedure)← Previous diff Revision as of 00:45, 30 January 2010 (view source) (→Procedure)Next diff → Line 6: Line 6: == Procedure == == Procedure == #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 #Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30. #Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30. #Make sure that the A260 measurement is between 0.1 and 1. #Make sure that the A260 measurement is between 0.1 and 1. #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 #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)]

## 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)]