Nanodrop

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 39: Line 39:
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:
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 <br>
dsDNA: 50 (uG/ml)-1 <br>
-
ssDNA or RNA: 38 (uG/ml)-1 <br>
+
ssDNA or RNA: 38 (uG/ml)-1
 +
*  Fasman, G.D. (Ed.) (1975) Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids, pp 589, 3rd edition, CRC Press.
 +
* Cantor C.R., Warshaw M.M., and Shapiro H. (1970) Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers, 9, 1059-1077.
 +
* Sprecher C.A., and Johnson W.C., Jr. (1977) Circular dichroism of the nucleic acid monomers. Biopolymers 16, 2243-2264.
 +
* Kallansrud G., and Ward B. (1996) A comparison of measured and calculated single- and double-stranded oligodeoxynucleotide extinction coefficients. Anal. Biochem. 236, 134-138.
 +
* Murphy J.H, and Trapane T.L. (1996) Concentration and extinction coefficient determination for oligonucleotides and analogs using a general phosphate analysis. Anal. Biochem. 240, 273-282.
 +
* Henderson J.T., Benight A.S., and Hanlon S. (1992) A semi-micromethod for the determination of the extinction coefficients of duplex and single-stranded DNA. Anal. Biochem. 201, 17-29.
 +
* Cavaluzzi, M.J., and Borer, P.N. (2004) Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 32, e13.
 +
 
===''Question''<br>===
===''Question''<br>===

Revision as of 13:59, 2 June 2005

Contents

Website

http://www.nanodrop.com/

FAQ

Question

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

Answer

From the nanodrop user manual (download at http://www.nanodrop.com/nd-1000-overview.html)

  • 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

  • Fasman, G.D. (Ed.) (1975) Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids, pp 589, 3rd edition, CRC Press.
  • Cantor C.R., Warshaw M.M., and Shapiro H. (1970) Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers, 9, 1059-1077.
  • Sprecher C.A., and Johnson W.C., Jr. (1977) Circular dichroism of the nucleic acid monomers. Biopolymers 16, 2243-2264.
  • Kallansrud G., and Ward B. (1996) A comparison of measured and calculated single- and double-stranded oligodeoxynucleotide extinction coefficients. Anal. Biochem. 236, 134-138.
  • Murphy J.H, and Trapane T.L. (1996) Concentration and extinction coefficient determination for oligonucleotides and analogs using a general phosphate analysis. Anal. Biochem. 240, 273-282.
  • Henderson J.T., Benight A.S., and Hanlon S. (1992) A semi-micromethod for the determination of the extinction coefficients of duplex and single-stranded DNA. Anal. Biochem. 201, 17-29.
  • Cavaluzzi, M.J., and Borer, P.N. (2004) Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 32, e13.


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?

Answer

This answer was received in an email response from an Application Scientist at Nanodrop Technologies, Inc.

"Yes, the Cell Cultures module is currently displayed at 1 mm and not 10 mm.

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.

Regarding microbial cell cultures in suspension:

The short answer: The ND-1000 can be used to estimate bacterial growth cultures, and any limitations will be similar to limitations of other spectrophotometers.

The long answer: The fundamental issue surrounding bacterial culture growth measurements is that an absorbance spectrophotometer is being asked to determine light scattering caused by particulates in suspension. In this case, transmittance is not related to absorbance in the classical sense. Under normal true absorbance conditions, spectrophotometers can be comparable to one another because the sample actually absorbs electromagnetic energy. In the case of a reduction of transmittance caused by light scattering, readings are very dependent on the optics of a specific spectrophotometer as well as the cell type in suspension. For bacterial growth determination, even 1cm path length systems can vary greatly due to the variability of the optics of each system. The ND-1000 will display an 'absorbance' value approximately 10 fold less than 1cm systems due to the fact that the instrument is utilizing a 1mm path length. However, the difference will not be exactly 10 fold due to the reasons I've described above. The point is that the differences between the ND-1000 and a 'conventional' spec (apart from the 10 fold difference due to path length), will be similar to differences found between spectrophotometers that utilize a 1cm path length."

Personal tools