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Desjardins P.R.,Thermo Scientific NanoDrop Products
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] | Year: 2011

Quantitation of DNA and RNA by absorbance and fluorescence spectroscopy has been a powerful tool in life sciences for decades. Classic methods of nucleic acid quantitation require the filling of devices, such as cuvettes and capillaries, with sample (traditional methodologies are described in APPENDIX 3D). Analysis of microvolume samples has become of paramount importance as more molecular biology techniques yield progressively smaller amounts of isolated sample and require accurate quantitation of nucleic acids with minimal consumption of sample. Advances in photonic technologies have resulted in a pioneering microvolume system that combines fiber optic technology with the inherent physical properties of the sample to dramatically reduce measurement volumes, removing the need for cuvettes and capillaries. Since the introduction of the first microvolume instrument, several new designs are now available, providing opportunities to measure nucleic acids using much smaller amounts of material. Altogether, these systems not only reduce measurement volume (as little as 0.5 to 2 μl), but also tend to be more efficient time-wise than traditional methods, making them useful even when sample is plentiful. The protocols in this unit are based on the most widely accepted microvolume systems and are intended as practical alternatives to traditional nucleic acid quantitation methodology. © 2011 by John Wiley & Sons, Inc. Source


Gallagher S.R.,UVP LLC | Desjardins P.,Thermo Scientific NanoDrop Products
Current Protocols in Essential Laboratory Techniques | Year: 2011

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. Two methods for direct absorbance measurements at 260 nm are described-the first is a traditional cuvette-based method, and the second is a microvolume method that requires no cuvettes or capillaries. In addition, three fluorescence techniques using Hoechst 33258, ethidium bromide, and PicoGreen reagent are presented in this unit. These five procedures cover a range from 5 to 10 ng/ml DNA to 15,000 μg/ml DNA. Reliable quantitation of proteins is possible using several types of assays. UV spectroscopy is the simplest approach but is limited in sensitivity. More sensitive assays that use Coomassie blue binding, bicinchoninic acid (BCA), and the Lowry reaction are also described. All assays are prone to amino acid composition errors and interference from assay solution components. Flow charts and tables to help with appropriate method selection are included. © 2011 by John Wiley & Sons, Inc. Source


Desjardins P.,Thermo Scientific NanoDrop Products
Journal of visualized experiments : JoVE | Year: 2010

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample. Source


Desjardins P.R.,Thermo Scientific NanoDrop Products | Conklin D.S.,Thermo Scientific NanoDrop Products
Current Protocols in Molecular Biology | Year: 2011

Quantitation of DNA and RNA by absorbance and fluorescence spectroscopy has been a powerful tool in life sciences for decades. Classic methods of nucleic acid quantitation require the filling of devices, such as cuvettes and capillaries, with sample (traditional methodologies are described in APPENDIX 3D). Analysis of microvolume samples has become of paramount importance as more molecular biology techniques yield progressively smaller amounts of isolated sample and require accurate quantitation of nucleic acids with minimal consumption of sample. Advances in photonic technologies have resulted in a pioneering microvolume system that combines fiber optic technology with the inherent physical properties of the sample to dramatically reduce measurement volumes, removing the need for cuvettes and capillaries. Since the introduction of the first microvolume instrument, several new designs are now available, providing opportunities to measure nucleic acids using much smaller amounts of material. Altogether, these systems not only reduce measurement volume (as little as 0.5 to 2 μl), but also tend to be more efficient time-wise than traditional methods, making them useful even when sample is plentiful. The protocols in this unit are based on the most widely accepted microvolume systems and are intended as practical alternatives to traditional nucleic acid quantitation methodology. © 2011 by John Wiley & Sons, Inc. Source


Desjardins P.,Thermo Scientific NanoDrop Products | Conklin D.,Thermo Scientific NanoDrop Products
Journal of Visualized Experiments | Year: 2010

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample. Copyright © 2010 Journal of Visualized Experiments. Source

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