DNA Quantification

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(Procedure)
(Procedure)
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.</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 23:45, 29 January 2010

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Contents

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)]

Notes

References

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