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