NanoDrop 1000 Software
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NanoDrop Product
Training
Topics
Computer Requirements
Nucleic Acid Module
A280 Module
MicroArray Module
Proteins and Labels Module
Colormetric Assays
Microbial Cell Culture
General UV/Vis
Data Viewer
Additional modules
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ComputerComputer
Requirements
Requirements
Microsoft XP or 2000 Operating System.
Windows Vista has also been tested successfully with NanoDrop software.
The operating software is not compatible with Windows NT, 95, 98 or ME.
Minimal requirements, no need for dedicated PC
NanoDrop 1000 and NanoDrop 3300
NanoDrop 8000
233 MHz or higher processor
800 MHz or higher processor
CD ROM drive
CD ROM drive
32 MB or more of RAM
128 MB or more of RAM
40 MB of free hard disk space
100 MB of free hard disk space
Open USB port (the instrument can only be connected via the USB port)
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Software Modules
Nucleic acid concentration and purity
(2.0ng/ul-3700ng/ul dsDNA)
Fluorescently labeled oligos for
microarray
Protein concentration (A280)
(0.1 mg/ml-100 mg/ml-BSA)
Fluorescently labeled proteins and
metalloproteins
Colorimetric protein assay
(i.e. Bradford, BCA, Lowry)
Microbial cell density measurements
General UV-Vis spectrophotometry
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Nucleic Acid Module
NanoDrop 1000 and NanoDrop 8000 Spectrophotometers
Used for Quality Control during Sample Preparation
 Nucleic acid quantitation and purity
 MicroArray probe preparation
 Quantitative RT-PCR
 Sequencing
 Genotyping
 Histocompatibility
 Microgenomics
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Nucleic Acid Module
Blank: Reference spectrum
NanoDrop 1000 interface
Re-blank: New reference spectrum as well
as display of last sample
Measure: Used to measure samples
Recording: Saves data to current report
Sample type: Color coded
Sample ID: Enter prior to sample
measurement. Changes through Data Viewer.
260/280 ratio: Sample purity indicator
260/230 ratio: Sample purity indicator
l: User selectable wavelength
10 mm path: Normalized
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Nucleic Acid Applications
Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifies cDNA following
its transcription from RNA and can be used when comparing
Different cell lines or tissues
Time course of drug treatment compared to the untreated control
Diseased versus nondiseased tissues
Critical that each reverse transcription reaction in the study contains equivalent amounts of RNA.
Laser capture microdissection (LCM) enables the isolation of desired pure cell
populations as limited as single cells from heterogeneous tissue samples.
Preserves essential cellular and morphological characteristics including the integrity of
biomolecules such as DNA, RNA, and proteins.
Often very low nucleic acid yield.
The time-limited nature of organ procurement and Human Leukocyte Antigens (HLA)
Typing requires instruments that are efficient as well as reliable
Bone marrow transplantation labs can have difficulty acquiring enough mononuclear
cells to get good DNA yields.
Dramatic acceptance of Nanodrop ND-1000 in HLA labs in 2 years.
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A280 Module
The A280 method is applicable to purified proteins exhibiting absorbance at 280nm.
The A280 Module does not
require generation of a standard
curve
Six sample type options
10 mm normalized path
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A280 Sample Type Options
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MicroArray Module
The capability to pre-select viable fluorescent-tagged hybridization probes for gene
expression in MicroArrays can eliminate potentially flawed samples and improve
research effectiveness.
Measures the concentration of
nucleic acid and the absorbance of
up to 2 fluorescent dyes.
Dye number selected using User
Preferences
Detects dye concentrations as low
as 0.2 picomole per microliter.
1 mm path
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Fluorescent Dyes
Fluorescent labels are useful to biomedical researchers running microarrays,
protein arrays, and flow cytometry.
Cy dyes are commonly used long-wavelength
dyes (Amersham)
Alexa Fluor dyes are generally more stable,
brighter, and less pH-sensitive than common dyes
(e.g. fluorescein, rhodamine) of comparable
excitation and emission. (Invitogen)
MicroArrays
Composed
of a collection of unique DNA probes
arranged on a solid substrate.
Probes composed of DNA sequences
complementary to the sequence of interest.
Nucleic acid “targets” incorporating fluorescent
dyes anneal to the complementary probes.
Differential color or signal intensity correlates with
target abundance.
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Proteins & Labels Module
This software module can be used to determine protein concentration
(A280nm) as well as up to 2 fluorescent dye concentrations
 l3: User selectable
Normalized to 10 mm path
Also used to measure the purity
of metalloproteins (such as
hemoglobin) using wavelength
ratios.
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Colormetric Assay Modules
Assay
Pierce BCA (Bicinchoninic
Acid)
20:1 reagent/sample volume
1:1 reagent/sample volume
Bradford (Coomassie)
50:1 reagent/sample volume
1:1 reagent/sample volume
Modified Lowry (Cupric
sulfate-tartrate)
Protein
Concentration
Range
0.2 to 8.0 mg/ml BSA
10 – 200 ug/ml BSA
0.1 to 8.0 mg/ml BSA
15 – 100 ug/ml BSA
0.2 – 4.0 mg/ml BSA
The Pierce BCA Assay is used for
more dilute protein solutions and/or
in the presence of components that
also have significant UV (280 nm)
absorbance.
Advantages
of Method
Disadvantages
of Method
Compatible with most
surfactants
Copper chelators, reducing
agents may interfere with the
BCA assay.
Fastest and easiest
protein assay.
Room temperature.
Linear range is 0.1-1
mg/ml
Surfactants may cause the
reagent to precipitate. Twice as
much protein-to-protein
variation as BCA assay.
“Un-Conditions” pedestals.
Can be measured at any
wavelength between 650
nm and 750 nm with little
loss of color intensity.
Detergents, potassium ions
form precipitates. Chelating
agents, reducing agents, and
free thiols interfere with this
assay.
The Bradford Assay response
varies with the composition of the
protein. The assay is also sensitive
to non protein sources, particularly
detergents, and becomes nonlinear
with higher protein concentrations.
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The Modified Lowry Protein Assay
Folin-Ciocalteu reagent is effectively
reduced in proportion to the chelated
copper-complexes .
Colormetric Modules
Tab structure to view
samples or standards
Valid only indicates
minimum number of
measurements made
Additional cursor position
available to measure
optional wavelength
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Colormetric Assay Standard Curves
Step 1: Measure the ‘Reference’
(Reagent only – a ‘zero’ Standard)
Step 2: Measure Standards
Up to 5 replicates each of up to 7
standards can be measured.
Step 3: Measure Samples
Sample concentrations can be
calculated by using linear interpolation
(point-to-point) between the two
standards flanking the unknown sample
or by using a polynomial fit.
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Colormetric Assay Standard Curves
NanoDrop Software offers Flexibility when using Standard Curves.
Curve Fit Options
Interpolation
Linear
2nd
or 3rd Polynomial
Save and Recall
Store
and reuse standard curve
NanoDrop 8000 allows for recall
of dilution concentration series
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Bradford vs BCA Results
Bradford Standard Curve
Pierce BCA Standard Curve
BCA is preferred when possible as better dynamic and linear range.
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Microbial Cell Cultures
Due to its shorter pathlength, the NanoDrop 1000 can measure cell densities that are
10-fold higher than those measurable on a standard cuvette spectrophotometer.
Diluted samples with low ‘Absorbance’
at 600 nm can be monitored at lower
wavelengths
(i.e. 320 nm)
Use 2 ul samples
Mix the culture well
Avoid bubbles
Measure quickly to avoid settling
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General UV- VIS
Displays absorbance measurements from 220 nm to 750 nm.
Has 2 cursors to permit measurement of individual peaks
User selectable baseline
User selectable normalization- lowest value 400-750 nm
Hi Abs feature- normalized to 0.1 nm on screen
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Additional Main Menu Options
Data Viewer
Account Management
User Preferences
Dye/Chromophore Editor
Utilities and Diagnostics
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Data Viewer
Data Viewer is a versatile, integrated data reporting software program
Offers the user the ability to customize report structures, import archived data and re-
plot data generated from NanoDrop instruments.
All data automatically archived on hard drive.
Accessed from either the Main Menu or the Show Report function
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Data Viewer
Re-plot Data
Import Archived Data
Customize Report Structures
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Utilities and Diagnostics
Selections
Calibration Check
Intensity Check
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Additional Main Menu Options
User Preferences
Account Management
Dye Chromophore/Editor
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NanoDrop 1000 FAQs
Q: What sort of accuracy and reproducibility, should I expect with the NanoDrop 1000?
A: Accuracy, typically within 2%. Reproducibility, typically +/- 0.003 A at low concentrations
Q: Is simply wiping the pedestal surface enough to prevent carryover?
A: Yes. The highly polished quartz and stainless steel surfaces of the sample retention system are
resistant to sample adherence, making the use of dry laboratory wipes very effective in removing the
sample.
Q: Do nucleic acids require purification prior to measurement on the NanoDrop 1000?
A: Yes. Absorbance measurements are not specific for a particular nucleic acid.
Q: Are there solvent restrictions?
A: Hydrofluoric acid can etch the quartz optical fiber. Most other laboratory solvents typically used in life
science labs, including dilute acids, are compatible as long as they are immediately wiped away.
Q: How do I check the accuracy of the NanoDrop 1000?
A: CF-1 calibration check fluid should be used with our Calibration Check module or software.
Q: How often do I need to check the accuracy of the NanoDrop 1000?
A: We recommend confirming that the instrument is within calibration specifications every 6 months
using the CF-1 Calibration Check Fluid .
Q: How long before I need to replace the flashlamp?
A: The lamp is rated to last for a minimum of 30,000 measurements before replacement could be
required.
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Sample Reproducibility
Use a 1.5-2 ul sample size
Erroneous results can occur when the liquid sample column is not completely
formed during a measurement. Note: Concentration calculations are volume
independent.
Ensure sample solution is homogeneous and purified
Important to ensure that the sample especially genomic DNA being measured
is homogeneous.
Confirm that your sample is within linear range of instrument
Measuring samples at or near the detection limit will result in higher CVs.
Confirm that the reference (blank) solution and sample solvent are the same
Buffers often absorb in the UV range.
Highly volatile solvents may not be conducive for use due to the rapid
evaporation and concentration of sample.
Use fresh aliquots for each measurement
Multiple measurements of the same aliquot will result in evaporation.
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