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
coupled device(CCD) array.

silicon

charge-

-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/L
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/molcm); 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/L)
*Procedure:
-Blanking cycle is performed , prior to running test
sample using the same solvent or buffer solution
that is present in the test samples.
-1-2l 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
100mg/ml(BSA)

purity

of

protein

up

to

-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
spectrophotometery.

visible

light-range

*Advantages:
-Requires very small sample volume(1-2L)
-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 microvolume sampling , heterogeneity will substantially
affect reproducibility.