# Difference between revisions of "Nanodrop"

## FAQ

### Question

How does the Nanodrop convert A260 to concentration in ng/uL?

• General

The Beer-Lambert equation is used to correlate the calculated absorbance with concentration:
A = E * b * c
Where A is the absorbance represented in absorbance units (A), E is the wavelength-dependent molar absorptivity coefficient (or extinction coefficient) with units of liter/mol-cm, b is the path length in cm, and c is the analyte concentration in moles/liter or molarity (M).

• For nucleic acid quantification, the Beer-Lambert equation is manipulated to give:

c = (A * e)/b
Where c is the nucleic acid concentration in ng/microliter, A is the absorbance in AU, e is the wavelength-dependent extinction coefficient in ng-cm/microliter and b is the path length in cm. The generally accepted extinction coefficients for nucleic acids are:
• Double-stranded DNA: 50
• Single-stranded DNA: 33
• RNA: 40
For the NanoDrop® ND-1000 Spectrophotometer,paths of 1.0 mm and 0.2 mm are used compared to a standard spectrophotometer using a 10.0 mm path. Thus, the NanoDrop® ND-1000 Spectrophotometer is capable of measuring samples that are 50 times more concentrated than can be measured in a standard spectrophotometer.

Note: absorbance data shown in archive files are represented as displayed on the software screen. For Nucleic Acid, Protein A280 and Proteins and Labels modules, data are normalized to a 1.0 cm (10.0 mm) path. For MicroArray, UV-Vis, Protein BCA, Protein Bradford, Protein Lowry and Cell Culture modules the data are normalized to a 0.1 cm (1.0 mm) path. For high absorbance UV-Vis samples, data are normalized to a 0.1mm path.

Other References

• The Biopolymer Calculator (http://paris.chem.yale.edu/extinct.html) calculates the extinction coefficient of nucleic acid sequences using an established base composition and nearest neighbor algorithm.

Comparing concentrations obtained with the calculated extinction coefficient vs. the nanodrop constant:
Calculated concentration was ~20% lower for two RNA sequences (subtillis pheB and BBa_I7101 mRNA) ~~cmc 11:13, 2 Jun 2005 (EDT)

• Molecular Cloning, Third Edition, A8.19 on Quantitation of Nucleic Acids:

Suggests that for short oligos it is important to calculate the extinction coefficient of a sequence of interest, but that for large molecules the following average extinction coefficients should be used: dsDNA: 50 (uG/ml)-1
ssDNA or RNA: 38 (uG/ml)-1

### Question

When you are measuring the concentration of DNA at 260 nm, the software automatically compensates for the fact that you are measuring your sample at a 1 mm path length instead of the standard 1 cm pathlength. For cell culture at OD 600 nm, the software does not do the same thing. Instead it displays the 1mm absorbance. Why?