DNA Quantification

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(Procedure)
(Procedure)
Line 5: Line 5:
== Procedure ==
== Procedure ==
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#Dilute the DNA sample 30X by combining the following in a cuvette
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1. 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.
+
2. Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30.
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#Make sure that the A260 measurement is between 0.1 and 1.
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3. Make sure that the A260 measurement is between 0.1 and 1.
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#If is too low then repeat the measurement using 15X dilution.
+
4. If is too low then repeat the measurement using 15X dilution.
:*84µl water
:*84µl water
:*6µl DNA prep
:*6µl DNA prep
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#Calculate the molar concentration of DNA using the following equation.</br>
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5. Calculate the molar concentration of DNA using the following equation:
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<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 00:47, 30 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

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

References

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