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NanoDrop Product Training
NanoDrop 1000 Software
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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
Topics
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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)
Computer RequirementsComputer Requirements
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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
Software Modules
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Nucleic acid quantitation and purity MicroArray probe preparation Quantitative RT-PCR Sequencing Genotyping Histocompatibility Microgenomics
Used for Quality Control during Sample Preparation
NanoDrop 1000 and NanoDrop 8000 Spectrophotometers
Nucleic Acid Module
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Nucleic Acid Module
Blank: Reference spectrum
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
: User selectable wavelength
10 mm path: Normalized
NanoDrop 1000 interface
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Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifies cDNA following its transcription from RNA and can be used when comparing
Different cell lines or tissuesTime course of drug treatment compared to the untreated control Diseased versus nondiseased tissuesCritical 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.
Nucleic Acid Applications
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The A280 Module does not require generation of a standard curve
Six sample type options
10 mm normalized path
The A280 method is applicable to purified proteins exhibiting absorbance at 280nm.
A280 Module
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A280 Sample Type Options
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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
MicroArray Module
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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)
MicroArraysComposed 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.
Fluorescent Dyes
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3: User selectable
Normalized to 10 mm path
Also used to measure the purity of metalloproteins (such as hemoglobin) using wavelength ratios.
This software module can be used to determine protein concentration (A280nm) as well as up to 2 fluorescent dye concentrations
Proteins & Labels Module
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AssayProtein
Concentration Range
Advantages of Method
Disadvantages of Method
Pierce BCA (Bicinchoninic Acid) 20:1 reagent/sample volume 1:1 reagent/sample volume
0.2 to 8.0 mg/ml BSA10 – 200 ug/ml BSA
Compatible with most surfactants
Copper chelators, reducing agents may interfere with the BCA assay.
Bradford (Coomassie)50:1 reagent/sample volume1:1 reagent/sample volume
0.1 to 8.0 mg/ml BSA15 – 100 ug/ml BSA
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.
Modified Lowry (Cupric sulfate-tartrate)
0.2 – 4.0 mg/ml BSA
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.
Colormetric Assay Modules
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.
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.
The Modified Lowry Protein Assay Folin-Ciocalteu reagent is effectively reduced in proportion to the chelated copper-complexes .
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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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Step 1: Measure the ‘Reference’ (Reagent only – a ‘zero’ Standard)
Step 2: Measure StandardsUp to 5 replicates each of up to 7 standards can be measured.
Step 3: Measure SamplesSample 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.
Colormetric Assay Standard Curves
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Curve Fit OptionsInterpolationLinear2nd or 3rd Polynomial
Save and RecallStore and reuse standard curveNanoDrop 8000 allows for recall
of dilution concentration series
NanoDrop Software offers Flexibility when using Standard Curves.
Colormetric Assay Standard Curves
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Bradford Standard Curve
Pierce BCA Standard Curve
Bradford vs BCA Results
BCA is preferred when possible as better dynamic and linear range.
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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 samplesMix the culture wellAvoid bubbles Measure quickly to avoid settling
Microbial Cell Cultures
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General UV- VIS
Displays absorbance measurements from 220 nm to 750 nm.Has 2 cursors to permit measurement of individual peaksUser selectable baselineUser selectable normalization- lowest value 400-750 nmHi Abs feature- normalized to 0.1 nm on screen
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Data Viewer
Account Management
User Preferences
Dye/Chromophore Editor
Utilities and Diagnostics
Additional Main Menu Options
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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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Customize Report Structures
Re-plot Data
Import Archived Data
Data Viewer
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Utilities and Diagnostics
Intensity Check Calibration Check
Selections
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Additional Main Menu Options
User Preferences Account Management
Dye Chromophore/Editor
25
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.
NanoDrop 1000 FAQs
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Use a 1.5-2 ul sample sizeErroneous 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 purifiedImportant to ensure that the sample especially genomic DNA being measured is homogeneous.
Confirm that your sample is within linear range of instrumentMeasuring 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 measurementMultiple measurements of the same aliquot will result in evaporation.
Sample Reproducibility