DNA Quantification

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Current revision (12:06, 14 March 2014) (view source)
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==References ==
==References ==
 +
"Convert ng to ug - Conversion of Measurement Units." http://www.convertunits.com/from/ng/to/ug
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]]
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]]

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Contents

Overview

This protocol uses a spectrophotometer to quantify the amount (μg/mL or ng/μL) 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 "enzymatic work" as digesting 500ng of a 4KB plasmid with the same multiple cloning site.

Procedure

1. Get DNA by any means necessary.
2. Run the DNA quantification (260/280) test on a spectrophotometer.

  • Be sure blank a sample first.
  • You can use only 1μL of sample if you use a NanoDrop
  • Or you can use 90μL of diluted sample using a UV cuvette
1. Dilute the DNA sample 30X by combining the following in a cuvette:
  • 87µl water
  • 3µl DNA prep
2. Run on the spectrophotometer with a dilution of 30.
3. 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

3. Calculate the molar concentration of DNA using the following equation:

  • Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]

4. Or calculate the μL of dna to add to obtain a desired molar amount of DNA.

  • μL = Picomoles*[0.66*DNA Size(bp)]/DNA Concentration(µg/ml)

Notes

  • One mole of single base pairs weighs 660 grams.
    • One picomole of 1000bp weighs 660ng.
  • 1ug = 1000ng
  • 0.001ug = 1ng

References

"Convert ng to ug - Conversion of Measurement Units." http://www.convertunits.com/from/ng/to/ug

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