DNA Quantification: Difference between revisions
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== 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 | |||
:*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 | |||
:*6µl DNA prep | |||
#Calculate the molar concentration of DNA using the following equation.</br> | #Calculate the molar concentration of DNA using the following equation.</br> | ||
*Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] | *Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] | ||
Revision as of 21:45, 29 January 2010
| back to protocols | ||
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
- Dilute the DNA sample 30X by combining the following in a cuvette
- 87µl water
- 3µl DNA prep
- 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.
- If is too low then repeat the measurement using 15X dilution.
- 84µl water
- 6µl DNA prep
- Calculate the molar concentration of DNA using the following equation.
- Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]
