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Nanodrop instrument

*General information:

-The NanoDrop is one of many spectrophotometer instruments

available from a range of manufactures , spectrophotometers are used in the quantification of nucleic acid ,protein and cell suspension concentrations as well as determination of DNA and protein purity.

-Models differ in terms of sample and range of linearity.

*Principle of operation:

-Light source: Xenon flash lamp.

-Detector:2048-element linear silicon charge-coupled device(CCD) array.

-Wavelength range:220-750nm.

-Absorbance of each sample is measured at two different path length (0.2nmm,1.0nm) allowing for a very wide range of detection (2-3700ng/µL dsDNA) without dilution.

-Absorbance calculation: Absorbance=-log(intensity [sample]/intensity [blank])

-Concentration calculation: *fluorescent dye concentration is calculated using the general form of the Berr-Lambert equation: A=Ebc, where A=absorbance ; E=extinction coefficient (liter/mol-cm); b=Path length, C=molarity.

*Nucleic acid concentration is calculated using a modified form of the Berr-Lambert equation:C=Ae/b , where e=extinction coefficient(ng-cm/µL)

*Procedure:

-Blanking cycle is performed , prior to running test sample using the same solvent or buffer solution that is present in the test samples.

-1-2µl of sample is pipeted on to a measurement pedestal which houses a fiber optic cable

-The sample arm is manually lowered over the sample

-Measurement is initiated by command from an attached PC

-After measurement is completed the sample arm is raised and surfaces that were in contact with the sample are wipe clean. The sample may be recovered with a pipet.

Nanodrop Spectrophotometer

*Applications:

-Measurement of nucleic acid concentration and purity

-Measurement of fluorescent dye labeling density of nucleic acid microarray samples.

-Analysis of the purity of protein up to 100mg/ml(BSA)

-Expanded spectrum measurement and quantitation of fluorescent dye-labeled proteins conjugates and metalloproteins

-Bradford assay analysis of protein

-Bicinchoninic acid(BCA) assay analysis of protein

-Lowry assay analysis of protein.

-Cell density measurements

-General UV and visible light-range spectrophotometery.

*Advantages:

-Requires very small sample volume(1-2µL)

-very wide dynamic range.

*Limitations:

-Micro-volume samples are subject to rapid evaporation; replicate measurement required reloading of fresh sample.

-There is risk of sample carryover if the instrument is not adequately cleaned between samples.

-DNA samples must be homogeneous due to micro-volume sampling , heterogeneity will substantially affect reproducibility.