NanoDrop 1000 Software

1

• NanoDrop 1000 Software

NanoDrop Product Training
2
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

3
Computer Requirements
Computer 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

4
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

5
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

6
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
• l User selectable wavelength
• 10 mm path Normalized

NanoDrop 1000 interface
7
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.

8
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

9
A280 Sample Type Options

10
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

11
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.

12
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.

13
Colormetric Assay Modules
Assay Protein Concentration Range Advantages of Method Disadvantages of Method
Pierce BCA (Bicinchoninic Acid) 201 reagent/sample volume 11 reagent/sample volume 0.2 to 8.0 mg/ml BSA 10 200 ug/ml BSA Compatible with most surfactants Copper chelators, reducing agents may interfere with the BCA assay.
Bradford (Coomassie) 501 reagent/sample volume 11 reagent/sample volume 0.1 to 8.0 mg/ml BSA 15 100 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.
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 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 Modified Lowry Protein Assay Folin-Ciocalteu
reagent is effectively reduced in proportion to
the chelated copper-complexes . 
14
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

15
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.
16
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

17
Bradford vs BCA Results
Bradford Standard Curve
Pierce BCA Standard Curve
BCA is preferred when possible as better dynamic
and linear range.
18
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  

19
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

20
Additional Main Menu Options

• Data Viewer
• Account Management
• User Preferences
• Dye/Chromophore Editor
• Utilities and Diagnostics

21
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

22
Data Viewer
Re-plot Data
Import Archived Data
Customize Report Structures
23
Utilities and Diagnostics
Selections
Calibration Check
Intensity Check
24
Additional Main Menu Options
User Preferences
Account Management
Dye Chromophore/Editor
25
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.
26
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.