Nanodrop

Posted on May 16th, 2016 by UNM CC

Measuring DNA concentration with the Nanodrop Spectrophotometer

v.1 10-31-2011
A. AlKhinji

Materials

  • DNA in TE buffer (10 mM Tris pH8, 1 mM EDTA)
  • TE buffer for blanking machine
  • P20 micropipettor + tips
  • H2O squeeze bottle and Kipwipes (at machine)

Procedure

  1. Sign log book
  2. Login to computer: (Adjacent lab P.I./room)
  3. Launch software: ND-100 v.3.7.1
  4. Wash the lower (sensor) pedestal and the upper (lid) pedestal with H2O spray bottle, then wipe both pedestals with a Kimwipe.
  5. Add 1-1.5 µL H2O. → Select "Initialize". Wipe pedestals clean.
  6. Add 1-1.5 λ TE → SELECT "Blank". Wipe pedestals.
  7. Repeat the following for each sample:
  8. Enter Sample #
  9. Add 1-1.5 µL DNA sample → Select "Measure".
  10. Wipe pedestals.
  11. Select "Show report" > Save report and export as Tab-delimited text file.
  12. When done: Wash the upper and lower pedestals with with H2O and wipe to dry.
  13. Select "Exit".
  14. Log off computer.

Comments

The Nanodrop will display an absorbance spectrum for each sample as it is being measured. DNA should have an absorbance peak centered at a wavelength of 260 nm (A260). The ratio A260/A280 should be ~1.95. The presence of organic solvents (e.g. phenol) may lead to a spuriously high A260/A280 ratio (> 2). Proteins will have a peak absorbance at 280 nm, so protein contamination will lower the A260/A280 ratio. Protein does not absorb as strongly as DNA so even a modest reduction in the A260/A280 ratio (e.g. 1.8) may represent a high level of contamination. The extinction coefficient is a factor that converts the peak absorbance to concentration. For DNA the extinction coefficient is 50 (ng/µL DNA) / A260. The nanodrop has a broad linear range. Accuracy drops off (error > 10%) for concentrations < 4 ng/µL and > 4000 ng/µL.

Tags: Fero-DNA

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