NanoDrop ND-1000 User manual

ND-1000 Spectrophotometer
V3.5User’s Manual
NanoDrop Technologies, Inc.
3411 Silverside Road
Bancroft Building
Wilmington, DE 19810 USA
Voice: 302-479-7707
Fax: [phone]
Email: [email protected]
www.nanodrop.com
NanoDrop is a registered trademark of NanoDrop Technologies, Inc. Other parties’
trademarks are the property of their respective owners and should be treated as such.
Copyright © 2007 NanoDrop Technologies, Inc.
rev 7/2007

Table of Contents
1. Overview ................................................................................................. 1-1
Instrument Description ............................................................................. 1-1
Operation ................................................................................................. 1-1
Applications.............................................................................................. 1-1
Patents..................................................................................................... 1-1
2. Initial Set Up ........................................................................................... 2-1
Computer Requirements.......................................................................... 2-1
Software Installation................................................................................. 2-1
Software Upgrades................................................................................... 2-2
Registering Your Instrument..................................................................... 2-2
3. General Operation.................................................................................. 3-1
The Sample Retention System................................................................. 3-1
Cleaning the Sample Retention System................................................... 3-1
Software Architecture and Features......................................................... 3-2
User Preferences ..................................................................................... 3-3
Utilities and Diagnostics........................................................................... 3-4
Account Management .............................................................................. 3-4
Dye/Chromophore Editor.......................................................................... 3-6
4. Common Module Functions.................................................................. 4-1
Module Startup......................................................................................... 4-1
Common Functions.................................................................................. 4-1
Measure (F1)............................................................................................ 4-1
Blank (F3)................................................................................................. 4-1
Re-blank (F2) ........................................................................................... 4-2
Print Screen (F4)...................................................................................... 4-2
Start Report / Recording........................................................................... 4-2
Print Report (F5) ...................................................................................... 4-3
Show Report (F7)..................................................................................... 4-3
Sample ID ................................................................................................ 4-3
Sample #.................................................................................................. 4-3
Exit........................................................................................................... 4-3
Escape Key (ESC) ................................................................................... 4-3
Show Context Help (Ctrl+H)..................................................................... 4-3
User’s Manual .......................................................................................... 4-3
5. Nucleic Acids.......................................................................................... 5-1
Sample Volume Requirements................................................................. 5-1
Measurement Concentration Range ........................................................ 5-1
Spectrum Normalization........................................................................... 5-2
Spectrum Overlay Control........................................................................ 5-2
6. MicroArray.............................................................................................. 6-1
Fluorescent Dye Selection ....................................................................... 6-1
Sample Volume Requirements................................................................. 6-1
Measurement Concentration Range ........................................................ 6-1
Baseline Calculation & Normalization....................................................... 6-2
7. UV-VIS..................................................................................................... 7-1
Sample Volume Requirements................................................................. 7-1
Measurement Concentration Range ........................................................ 7-1
Unique Screen Features .......................................................................... 7-1
8. Protein A280 ........................................................................................... 8-1
Sample Volume Requirements................................................................. 8-1
Pedestal Reconditioning........................................................................... 8-1
Measurement Concentration Range ........................................................ 8-1
Unique Screen Features .......................................................................... 8-1
Spectrum Normalization........................................................................... 8-2
Spectrum Overlay Control........................................................................ 8-3
9. Proteins & Labels................................................................................... 9-1
Fluorescent Dye Selection ....................................................................... 9-1
Sample Volume Requirements................................................................. 9-1
Pedestal Reconditioning........................................................................... 9-1

Measurement Concentration Range ........................................................ 9-1
Unique Screen Features .......................................................................... 9-2
Baseline Type .......................................................................................... 9-2
10. Protein BCA.......................................................................................... 10-1
Sample Volume Requirements............................................................... 10-1
Pedestal Reconditioning......................................................................... 10-1
Measurement Concentration Range ...................................................... 10-1
BCA Kits, Protocols, and Sample Preparation ....................................... 10-1
Unique Screen Features ........................................................................ 10-1
Making BCA Measurements................................................................... 10-2
Standard Curve Features....................................................................... 10-3
Delete Standard Points .......................................................................... 10-3
Exiting the BCA Module ......................................................................... 10-4
11. Protein Lowry ....................................................................................... 11-1
Sample Volume Requirements............................................................... 11-1
Pedestal Reconditioning......................................................................... 11-1
Measurement Concentration Range ...................................................... 11-1
Modified Lowry Kits, Protocols, and Sample Preparation....................... 11-1
Unique Screen Features ........................................................................ 11-1
Making Lowry Measurements ................................................................ 11-2
Standard Curve Features....................................................................... 11-3
Delete Standard Points .......................................................................... 11-3
Exiting the Lowry Module ....................................................................... 11-4
12. Protein Bradford................................................................................... 12-1
Sample Volume Requirement ................................................................ 12-1
Pedestal Reconditioning......................................................................... 12-1
Measurement Concentration Range ...................................................... 12-1
Bradford Kits, Protocols, and Sample Preparation................................. 12-1
Unique Screen Features ........................................................................ 12-2
Making Bradford Protein Measurements................................................ 12-2
Standard Curve Features....................................................................... 12-3
Delete Standard Points .......................................................................... 12-3
Exiting the Bradford Module................................................................... 12-4
13. Cell Cultures......................................................................................... 13-1
Sample Size Requirements.................................................................... 13-1
Cell Suspension Concentrations ............................................................ 13-1
Sample Homogeneity............................................................................. 13-2
Decontamination of Measurement Pedestals......................................... 13-2
14. Archived Data and Data Viewer .......................................................... 14-1
Archive File Creation.............................................................................. 14-1
Data Storage Hierarchy.......................................................................... 14-1
Data Viewer............................................................................................ 14-2
Archive File Converter............................................................................ 14-5
Opening Archived Data with Spreadsheet Programs............................. 14-6
15. Calibration Check................................................................................. 15-1
Procedure............................................................................................... 15-1
16. Troubleshooting................................................................................... 16-1
Error USB2000....................................................................................... 16-1
Connection Error.................................................................................... 16-2
Signal Error ............................................................................................ 16-2
Saturated Detector................................................................................. 16-3
Liquid Column Breakage........................................................................ 16-3
Other Software Error Messages............................................................. 16-5
Sample Accuracy and Reproducibility.................................................... 16-6
260/280 Ratio......................................................................................... 16-7
Unusual Spectrum.................................................................................. 16-7
Technical Service................................................................................... 16-8
17. Maintenance and Warranty.................................................................. 17-1
Cleaning................................................................................................. 17-1
Calibration.............................................................................................. 17-1
Warranty................................................................................................. 17-1
18. Appendices........................................................................................... 18-1
Instrument Specifications....................................................................... 18-1
Blanking and Absorbance Calculations.................................................. 18-1

Concentration Calculation (Beer’s Law)................................................. 18-1
Solvent Compatibility.............................................................................. 18-2
Decontamination of Measurement & Optical Surfaces........................... 18-2
Setting Up a Dymo 400 Label Writer Printer .......................................... 18-2

Section 1- Overview
1. Overview
Instrument Description
The NanoDrop®ND-1000 is a full-spectrum (220-750nm) spectrophotometer that measures 1 ul samples with high
accuracy and reproducibility. It utilizes a patented sample retention technology that employs surface tension alone to hold
the sample in place. This eliminates the need for cumbersome cuvettes and other sample containment devices and
allows for clean up in seconds. In addition, the ND-1000 has the capability to measure highly concentrated samples
without dilution (50X higher concentration than the samples measured by a standard cuvette spectrophotometer).
Operation
A 1 ul sample is pipetted onto the end of a fiber optic cable (the receiving fiber). A second fiber optic cable (the source
fiber) is then brought into contact with the liquid sample causing the liquid to bridge the gap between the fiber optic ends.
The gap is controlled to both 1mm and 0.2 mm paths. A pulsed xenon flash lamp provides the light source and a
spectrometer utilizing a linear CCD array is used to analyze the light after passing through the sample. The instrument is
controlled by special software run from a PC, and the data is logged in an archive file on the PC.
Applications
UV/VIS spectrophotometry is simple for samples as small as 1 ul using the NanoDrop®ND-1000 Spectrophotometer. The
small sample requirement and ease of use make the NanoDrop®ND-1000 Spectrophotometer ideally suited for
measuring:
•Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng/ul (dsDNA) without dilution
•Fluorescent dye labeling density of nucleic acid microarray samples
•Purified protein analysis (A280) up to 100 mg/ml (BSA)
•Expanded spectrum measurement and quantitation of fluorescent dye labeled proteins, conjugates, and
metalloproteins
•Bradford Assay analysis of protein
•BCA Assay analysis of protein
•Lowry Assay analysis of protein
•Cell density measurements
•General UV-Vis spectrophotometry
Patents
The sample retention technology used in the ND-1000 is covered under US patents 6,628,382 and 6,809,826. Other
patents are pending.
1-1

Section 2- Initial Set Up
2. Initial Set Up
Computer Requirements
The NanoDrop software will only run on an IBM compatible PC meeting the below criteria.
•Microsoft Windows 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.
•233 MHz or higher processor
•CD ROM drive
•32 MB or more of RAM
•40 MB of free hard disk space
•Open USB port (the instrument can only be connected via the USB port)
•Microsoft Excel or other spreadsheet program to manipulate archived data (optional)
Software Installation
WARNING: The system software must be loaded onto the PC before the USB cable is connected. Administrator
access on the PC is required to install the software.
To properly install NanoDrop software:
1.Close all programs and make sure that the USB cable is unplugged.
2.Insert the operating software CD in the CD drive of the PC. The software installation menu should appear
automatically. If the software menu does not appear, choose ‘My Computer‘ to view the contents of the CD. Double
click on the file named ‘nd-1000…install.exe’.
3.After software installation, connect the USB cable and the Found New Hardware Wizard should start as shown below.
(Windows XP SP2 operating system will ask to allow it to search the internet for the proper software as shown- Select
‘No, not this time’). Follow the prompts for automatic installation of the software.
Intro Page: Windows XP- SP2 All Windows Operating
Systems
Your NanoDrop®ND-1000 Spectrophotometer should now be ready for operation. If the software does not start properly,
refer to the “Troubleshooting” section for possible solutions.
Configuring the System Font
The NanoDrop software is designed to look best with the MS Sans Serif font, 8 point. To check that the system font is set
to the proper selection:
1.Open the ‘Displays Properties’ by right clicking on the desktop and select Properties
Æ Appearance. (Additional step
for Windows XP: click on the ‘Advanced’ button).
2.From ‘item’ list select ‘icon’.
3.Select the ‘MS Sans Serif (western)’ font and select ‘8 point’ size.
4.Click OK.
Other selections can be used, but may either cause some text in the NanoDrop software window to not fit well or result in
the function selection tabs across the top to become inaccessible.
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Section 2- Initial Set Up
Software Upgrades
NanoDrop Technologies makes periodic upgrades to the NanoDrop software. These upgrades are available for download
at www.nanodrop.com.
Cable Connections
To make measurements with the instrument, connect the USB cable to instrument and the PC, plug in the 12V power
supply and connect to the power input at the back of the instrument.
Note: The power supply can remain plugged into the NanoDrop®ND-1000 Spectrophotometer while the instrument is not
in use. When the unit is in this “standby” mode, power consumption is ~1.5 W and the flashlamp is not energized. Also,
the instrument does not utilize a power switch or give a visual indication of the operability of the 12V power supply.
Registering Your Instrument
Please register your product! We periodically update our software and add new features free of charge. We would like to
keep our user list updated so that we may alert you to these updates. All information supplied to NanoDrop Technologies
is completely confidential. You can register at www.nanodrop.com.
2-2

Section 3-General Operation
3. General Operation
The Sample Retention System
Basic Use
The main steps for using the sample retention system are listed below:
1. With the sampling arm open, pipette the sample onto the lower
measurement pedestal.
2. Close the sampling arm and
initiate a spectral measurement
using the operating software on
the PC. The sample column is
automatically drawn between the
upper and lower measurement
pedestals and the spectral
measurement made.
3. When the measurement is
complete, open the sampling arm
and wipe the sample from both
the upper and lower pedestals
using a soft laboratory wipe.
Simple wiping prevents sample
carryover in successive
measurements for samples
varying by more than 1000 fold in
concentration. See
www.nanodrop.com for
performance data on sample
carryover.
Cleaning the Sample Retention System
Wiping the sample from both the upper and lower pedestals (as shown above) upon completion of each sample
measurement is usually sufficient to prevent sample carryover and avoid residue buildup. Although generally not
necessary, 2 ul water aliquots can be used to clean the measurement surfaces after particularly high concentration
samples to ensure no residual sample is retained on either pedestal. After measuring a large number of samples,
however, it is recommended that the areas around the upper and lower pedestals be cleaned thoroughly. This will prevent
the wiping after each measurement from carrying previous samples onto the measurement pedestals and affecting low-
level measurements. A final cleaning of all surfaces with de-ionized water is also recommended after the user’s last
measurement. Note: Please do not use a squirt bottle to apply de-ionized water.
Decontamination of Measurement Pedestals
If decontamination is necessary, a sanitizing solution, such as a 0.5% solution of sodium hypochlorite (1:10 dilution of
common commercial bleach solutions – freshly prepared), can be used to ensure that no biologically active material is
3-1

Section 3-General Operation
present on the measurement pedestals. The metal fiber optic fittings are made from 303 stainless steel and are resistant
to most common laboratory solvents (see “Solvent Compatibility” appendix). Note: Please do not use a squirt bottle to
apply diluted bleach.
Special Cleaning Requirements for Proteins
Proteins and solutions containing surfactants can “un-condition” the measurement pedestal surfaces so that the liquid
column does not form well with 1ul samples. If this occurs, “buff” the measurement pedestal surfaces by rubbing each
measurement surface aggressively with a dry laboratory wipe 30-40 times. This will “re-condition” the surface allowing the
liquid sample column to form. Alternatively, use the NanoDrop Pedestal Reconditioning Compound (PR-1) as a rapid
means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break
during measurement. Additional information about the PR-1 kit may be found at www.nanodrop.com.
Sample Size Requirements
Although sample size is not critical, it is essential that the liquid column be formed so that the gap between the upper and
lower measurement pedestals is bridged with sample.
Field experience indicates that the following volumes are sufficient to ensure reproducibility:
•Aqueous solutions of nucleic acids: 1 ul
•Purified protein: 2 ul
•Bradford, BCA or Lowry assay: 2 ul
•Microbial cell suspensions: 1-2 ul
It is best to use a precision pipettor (0-2 ul) with precision tips to assure that sufficient sample (1-2 ul) is used. Lower
precision pipettors (0-10 ul and larger) are not as good at delivering 1 ul volumes to the measurement pedestal. If you are
unsure about your sample characteristics or pipettor accuracy, a 2 ul sample is recommended.
Sample Carryover
Prevention of sample being retained on the ND-1000 Spectrophotometer’s measurement pedestals is easily addressed.
Simple wiping of the upper and lower measurement pedestal with a dry laboratory wipe is highly effective in eliminating
carryover for samples differing in concentration by as much as three orders of magnitude (see data at
www.nanodrop.com). This is possible since each measurement pedestal is in actuality a highly polished end of a fiber
optic cable. There are no cracks or crevices for residual sample to get trapped within.
Sample Homogeneity
Sampling from non-homogeneous solutions – particularly when using small volumes – can cause significant deviations in
the data generated using all measurement technologies including spectrophotometry. Genomic DNA, lambda DNA and
viscous solutions of highly concentrated nucleic acids are common examples known to the molecular biologist. Proteins
are subject to denaturation, precipitation, and aggregation and therefore may require special handling to ensure sample
homogeneity.
Effect of Evaporation and Solvents
Evaporation of the sample during the measurement cycle usually has just a minimal effect on absorbance readings and
may result in a 1-2% increase in sample concentration. This can be observed in the field by measuring the same sample
successively over time. Highly volatile solvents, such as hexane, will likely evaporate before the measurement can be
completed. Less volatile solvents such as DMSO can be used successfully.
Sample Recovery
One of the advantages of the sample retention system is that samples can be recovered from the upper and lower
measurement pedestals by extraction with a pipette.
Software Architecture and Features
Main Menu
With the sampling arm in the down position, start the NanoDropsoftware by selecting the following path:
Start
Æ Programs Æ NanoDrop Æ ND-1000 (version)
3-2

Section 3-General Operation
Application Modules
The NanoDrop software has been tailored to meet the life scientist’s needs. It includes the following application modules:
•Nucleic Acid – concentration and purity of nucleic acid
•MicroArray – dye incorporation concentration and purity of nucleic acid
•UV-Vis – general UV-Vis measurements
•Cell Cultures – “absorbance” (light scattering) measurement of suspended microbial cells
•Protein A280 – concentration and purity of purified protein
•Proteins & Labels – concentration of dye-labeled proteins, conjugates, and metalloproteins
•Protein BCA – protein concentration using the BCA assay
•Protein Bradford – protein concentration using the Bradford assay
•Protein Lowry – protein concentration using the Modified Lowry assay
User Preferences
Each user has the option to configure a number of settings in the various application modules. Some key preference
options available for each of the User Preference tabs are as follows
•Archiving
In addition to the primary data storage of archive files at c:\nanodrop data, users may elect to save their data to an
additional location. This option can be chosen under the ‘Archiving.’ tab by selecting the ‘Duplicate data storage?’ box
and then choosing the file path by clicking on the file folder icon under ‘Duplicate Data Folder’. Save the alternative
path by clicking on the ‘Save and Exit’ button before exiting the User Preferences module. The user may also elect to
deselect an automatic prompt to close the Data Viewer whenever a module is closed. The Data Viewer must be
closed if a different module is opened before data can be reviewed.
•Reports
Users may choose to select the ‘Auto Reporting’ option for any of the application modules. The auto reporting option
allows data to automatically be saved to the report for all samples. Users may choose this option under the Report tab
by selecting the corresponding box next to the modules listed under ‘Auto Reporting’. Save the auto reporting
functions by clicking on the ‘Save Preferences’ button before exiting the User Preferences window.
Note: User preferences are stored in a ‘.log’ file. When upgrading to a newer version of the software, this file should be
preserved. If after upgrading to a new software version the user preferences do not appear correctly, the .log file
should be manually copied to the proper directory. See “Passwords. log’” for more detail.
•Nucleic Acids
The default setting is DNA-50. Other options include RNA-40, ssDNA-33, and Other with a variable constant setting.
•UV/Vis
The default settings for the two cursors used to monitor specific wavelengths are 300 nm for λ1 and 700 nm for λ2. The
user may elect to have the HiAbs on (automatic utilization of the 0.2 mm path). An additional option is to elect to
3-3

Section 3-General Operation
normalize the data and spectra using the absorbance value of the wavelength between 400 nm and 700 with the
lowest absorbance value.
•Microarray
The default setting is ssDNA-33 for the nucleic acid. The default setting from NanoDrop remain Dye 1 set to Cy3 with
absorbance normalized to the absorbance value at 750nm.
Other options include RNA-40, ssDNA-33, Other with several hard-coded dye choices including common Alexa fluor
dyes. See the section on Dye/Chromophore Editor at the end of chapter 4 for more information.
•A280
There are six sample types (options) available for purified protein analysis and concentration measurement. The
default setting is Other protein (E1%). See Section 8 for additional information about each sample type option. Note:
New to version 3.5.1 is the ability to select whether or not to have the data and spectrum normalized to the absorbance
value at 340 nm.
•Proteins and Labels
There are six sample types (options) available for purified protein analysis and concentration measurement. The
default setting is Other protein (E1%).
The user may also use elect whether or not to use a bichromatic normalization to the absorbance value at 340 nm. The
default setting from NanoDrop remains Dye 1 set to Cy3 with absorbance normalized at 750nm.
Utilities and Diagnostics
This module is used to both confirm that the instrument is performing within the pathlength calibration specifications and
help troubleshoot operational problems with the instrument. Note: The inclusion of the calibration check utility is new to
version 3.5.1.
For more information on using this module, refer to both section 15 (Calibration Check) and section 16 (Troubleshooting)
of this manual.
Account Management
The Account Management module provides options for directing where specific data files are archived by allowing users to
segregate their data into personal folders. The Account Management module is accessible to the administrator only.
Account Types
There are three types of user accounts:
•Level 10- this is the highest security setting and all level 10 users can add new users, modify a user, delete a user and
set password options. At the time of software installation, the only level 10 account is Administrator whose initial
password is “nanodrop”. It is strongly recommended that the password be changed after initial account set up. Any
user can be set to a level 10 access, although this is not recommended (see Level 5). Note: The administrator (or the
last level 10 user) account may not be deleted.
•Level 5- this is the security setting recommended for an ordinary user account. An account with this access will be
password protected and will be able to select specific user preferences. Also, all data generated will be automatically
archived to the user’s account in c:\nanodrop data (and the user specified location if that preference is selected).
3-4

Section 3-General Operation
•Default (level 0 security) - this access level is reserved for the Default account only. This account enables any user
without an account to access all the active software measurement modules. Although it is not password protected, user
preferences can be set for this account. All data generated will be automatically archived to the Default folder within the
c:\Nanodrop Data folder. Note: For laboratories requiring that every user have a unique user-account, the administrator
may disable the default user account.
Account Log-in/Log-out and Time Out
The user’s account will remain active until 1) a user logs out of his/her account by using the pull down menu to select
either Default or another user name or 2) the user closes the software.
A user account may also be logged out automatically if the software “System Idle Timeout” is exceeded. After 4 hours of
inactivity the software account will automatically revert back to the Default user. A screen will appear indicating that the
time is about to expire, with a 30-second countdown. If the user elects ‘CANCEL’, the clock with reset and the user
account and application module will remain active for another 4 hours. If the time expires, the open application module will
close, returning to the Main Menu and the Default user.
Account Lockout
User-specific accounts can become locked out in several ways as noted below:
•Failure to change password within the allotted time
•Incorrectly entering the password 99 consecutive times
•The administrator locks a specific account
Only the administrator (level 10) can unlock a locked account. This is done by using the ‘Modify User’ entry in the Account
Management module. Note: All accounts (even the administrator) can be locked if the incorrect password entry occurs as
previously described.
Change Password
This module enables each user having an authorized account ID to change their respective password.
Note: The administrator, using the ‘Options’ or the ‘Modify User’ entries in the ‘Account Management’ module, establishes
whether individual user passwords will expire and, if so, after how many days.
Passwords.log file
This file contains the User ID & password for all accounts and is readable only by the software. It can be found in the
c:\nanodrop data\log files folder. It is strongly recommended that the administrator make a copy of that file and store it in
the same log files folder as above each time a new user account is added or a password is changed. If the administrator’s
account becomes locked, the up-to-date copy can be renamed and used as the password.log file.
Note: If upgrading from a previous version, the “passwords.log” and “user preferences.log” files should be automatically
copied to the c:\NanoDrop Data\Log Files directory. If for some reason these files are not copied automatically, they must
be manually copied from the c:\program files\NanoDrop (version) to the c:\program files\NanoDrop (version) directory.
3-5

Section 3-General Operation
Dye/Chromophore Editor
The Dye/Chromophore Editor gives the user the ability to add their own dyes or chromophores in addition to the
predefined fluorescent dyes available for use with the MicroArray and Proteins and Labels modules. Note 1: Predefined
dye methods are indicated by a diamond and can’t be modified. Note 2: Absorbance contribution at 260 nm and 280 nm
from the respective dye can be corrected by entering the appropriate decimal correction % field. Refer to the dye
manufacturer to find the 260 nm and 280 nm % factor for dyes not pre-defined in the Dye/Chromophore List.
Note: If upgrading from a version prior to 3.3, zero values (0) for 260 nm % and 280 nm % correction factors will be
entered for all user defined dyes.
3-6

Section 4-Common Module Functions
4. Common Module Functions
Module Startup
When the software starts, you should see this message:
For best results, ensure measurement pedestal surfaces are clean and load a water sample onto the lower
measurement pedestal and then click ‘OK’. After clicking OK, the message “Initializing Spectrometer- please wait” will
appear. When this message disappears, the instrument will be ready for use. All data taken will automatically be logged
in the appropriate archive file.
Common Functions
Measure (F1)
Each time a software module is opened (initiated), the Measure’ button is inactive as noted by its “grayed-out”
appearance. A blank must first be measured before the Measure button will become active.
The Measure button is used to initiate the measurement sequence for all samples (non-blanks). It is actuated by
depressing the F1 key or clicking the ‘Measure’ button. The entire measurement cycle takes approximately 10 seconds.
Blank (F3)
Before making a sample measurement, a blank must be measured and stored (see “Blanking and Absorbance
Calculations” in the appendix for more details on absorbance calculations). After making an initial blank measurement, a
straight line will appear on the screen; subsequent blanks will clear any sample spectrum and display a straight line as
shown in the following image:
For the most consistent results, it is best to begin any measurement session with a blanking cycle. This will assure the
user that the instrument is working well and that the pedestal is clean. To perform a blanking cycle, perform the following:
1.Load a blank sample (the buffer, solvent, or carrier liquid used with your samples) onto the lower measurement
pedestal and lower the sampling arm into the ‘down’ position.
2.Click on the ‘Blank’ (F3) button.
3.When the measurement is complete,wipe the blanking buffer from both pedestals using a laboratory wipe.
4-1

Section 4-Common Module Functions
4.Analyze an aliquot of the blanking solution as though it were a sample. This is done using the ‘Measure’ button (F1).
The result should be a spectrum with a relatively flat baseline. Wipe the blank from both measurement pedestal
surfaces and repeat the process until the spectrum is flat.
See “Blanking and Absorbance Calculations” in the appendix for more information on blanking and absorbance
calculations.
Re-blank (F2)
The Re-blanking option (F2) establishes a new reference (blank) that is used for the absorbance calculations of
subsequent samples. However, unlike the Blank (F3) function, the Re-blank feature recalculates the absorbance
spectrum for the most recent sample and displays this on the screen. When the Re-blank function is used, the following
message appears:
Print Screen (F4)
The ‘Print Screen’ button will print a copy of the current operating screen to the default printer attached to the operating
PC.
Note: The system is configured to work with the Dymo Label Writer 400 printing on #30256 [2-5/16” X 4”] shipping labels,
but can print on any printer connected to the PC.
Print Window
A Print dialogue can be initiated from the ‘File’ pull-down menu or by typing ‘Ctrl+P’. The user can specify any connected
printer to print to from the Print dialogue.
Saving Current Screen as .JPG Image
The current screen can be saved as a .jpg image file by selecting ‘Save Window’ from the ‘File’ pull down menu.
Start Report / Recording
The user can log measurement results in a report table and print them to the desired printer. To initiate this feature, select
the ‘Start Report’ button. The default setting has the Recording feature activated. Refer to section 14 (Data Viewer) for
additional details. Note: To override this feature, click on the ‘Recording’ button. Once de-selected, the button will read
Start Report.
When the specified maximum number of entries for that specific report has been reached, there are 4 options: ‘Ignore’, ‘Save’, ‘Print’,
‘Save and Print’.
All data is stored in the archive file at c:\NanoDrop Data (and in a duplicate location if selected in User Preferences).
4-2

Section 4-Common Module Functions
Note: This feature can be set so that ‘Recording’ is the default mode. See “User Preferences” in section 3 for more
information.
Print Report (F5)
Selecting the ‘Print Report’ (F5) button will print the existing sample report to the default printer. It can be configured to
clear the sample report contents. The user also has options as to how the buffer is handled. Refer to section 14 (Data
Viewer) for additional details. All data is stored in the archive file at c:\NanoDrop Data and in a duplicate location if
selected in User Preferences.
Note: The system is configured to work with the Dymo Label Writer 400 printing on #30256 [2-5/16” X 4”] shipping labels,
but can print to any printer connected to the PC.
Show Report (F7)
The user can display the entries comprising the current Sample Report at any time by selecting the ‘Show Report’ button.
This function will enable the Data Viewer software described in section 14. Parameters specific for the individual
application modules are populated for each individual Sample ID.
Sample ID
The ‘Sample ID’ is highlighted for overtyping or barcode scanning. The user may input a sample ID that will be used to
identify the measurement in a report print and in the archived data file. The sample ID entry is “key focused”, meaning it is
the default selection on the screen and should have a flashing text cursor when the instrument is waiting to make a new
measurement.
Sample #
The ‘Sample #’ indicator is activated when a sample report is being recorded. It indicates the sample number of the last
sample processed in the current report and increments with each successive measurement until the sample report is fully
populated. The sample buffer limit can be modified on the report page.
Exit
This command closes all application modules and supporting options. After clicking the ‘Exit’ button, the user has 10
seconds to cancel the exit command. If no action is taken within 10 seconds, the exit command is carried out. Note: All
measurement data is automatically saved to an archive file and requires no user action.
Escape Key (ESC)
The escape key is set to exit out of all screens. Hitting the escape key twice will log the user out of an application module.
Show Context Help (Ctrl+H)
Context Help is enabled in the Main Menu, all function modules, and the application modules. The help feature is enabled
by choosing ‘Show Context Help’ from the ‘Help’ menu pull down or by selecting ‘Ctrl+H’. Once enabled, placing the
cursor on elements of the screen will automatically generate an explanation of that element. Context Help remains active
until deselected.
User’s Manual
A .PDF version of this User’s Manual is accessible from the Main Menu and from the Help menu in all of the application
modules. It can also be accessed by selecting from the Help pull down menu in any application module or from Start
Æ
Programs
Æ NanoDrop Æ ND-1000 (version).
4-3

Section 5- Nucleic Acids
5. Nucleic Acids
Nucleic acid samples can be readily checked for concentration and quality using the NanoDrop®ND-1000
Spectrophotometer. To measure nucleic acid samples select the ‘Nucleic Acid’ application module.
Sample Volume Requirements
Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when
measuring aqueous nucleic acid samples. However, if you are unsure about your sample or your pipettor accuracy, a 1.5-
2ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by
sample.
Measurement Concentration Range
The NanoDrop® ND-1000 Spectrophotometer will accurately measure dsDNA samples up to 3700 ng/ul without dilution.
To do this, the instrument automatically detects the high concentration and utilizes the 0.2mm pathlength to calculate the
absorbance.
Detection
Limit
(ng/ul)
Approx.
Upper Limit
(ng/ul)
Typical Reproducibility
(minimum 5 replicates)
(SD= ng/ul; CV= %)
2 3700 ng/ul (dsDNA)
3000 (RNA)
2400 (ssDNA)
sample range 2-100 ng/ul: ±2 ng/ul
sample range >100 ng/ul: ±2%
Unique Screen Features
Sample Type: used to select the (color-keyed) type of nucleic acid being measured. The user can select ‘DNA-50’ for
dsDNA, ‘RNA-40’ for RNA,‘ssDNA-33’ for single-stranded DNA, or ‘Other’ for other nucleic acids. The default is DNA-50.
If ‘Other’ is selected, the user can select an analysis constant between15-150. When navigating amongst the three
general sample types within the Nucleic Acids module, the last constant value entered within the ‘Constant’ sample type
will be retained. See the “Concentration Calculation (Beer’s Law)” Appendix for more details on this calculation.
λand Abs: the user selected wavelength and corresponding absorbance. The wavelength can be selected by moving the
cursor or using the up/down arrows to the right of the wavelength box. Note: The user-selected wavelength and
absorbance are not utilized in any calculations.
A260 10 mm path: absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm path.
Note: This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually
measured using the 0.2 mm path length.
A280 10 mm path: sample absorbance at 280 nm represented as if measured with a conventional 10 mm path. Note:
This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured
using the 0.2 mm path length.
5-1

Section 5- Nucleic Acids
260/280: ratio of sample absorbance at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess
the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted
as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other
contaminants that absorb strongly at or near 280 nm. See “260/280 Ratio” section of the Troubleshooting section for more
details on factors that can affect this ratio.
260/230: ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purity. The
260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. They are commonly in the
range of 1.8-2.2. If the ratio is appreciably lower, this may indicate the presence of co-purified contaminants.
ng/ul: sample concentration in ng/ul based on absorbance at 260 nm and the selected analysis constant. See the
“Concentration Calculation (Beer’s Law)” in the appendix for more details on this calculation.
Spectrum Normalization
The baseline is automatically set to the absorbance value of the sample at 340 nm, which should be very nearly zero
absorbance. All spectra are referenced off of this zero.
Spectrum Overlay Control
The user can display more than one spectrum in the same display using this feature. The current sample plot will be
displayed in bold and previous plots will be distinguished by different colors as seen in the following example:
The default option is set to clear the display for the next reading. The user may set the overlay control to clear after each
sample plot, (default setting), after each new report, or accumulate plots until prompted to clear. The ‘Clear Now’ setting
will clear all current and previous plots. When the overlay function is active, the software will auto scale the y-axis based
on the sample with the highest absorbance at 260 nm. Note: When the overlay function is active, the ‘Blank’ function does
not clear the existing overlaid sample spectra.
5-2

Section 6- MicroArray
6. MicroArray
The capability to pre-select viable fluorescent-tagged hybridization probes for gene expression in micro arrays can
eliminate potentially flawed samples and improve research effectiveness. The NanoDrop® ND-1000 Spectrophotometer
measures the absorbance of the fluorescent dye, allowing detection at dye concentrations as low as 0.2 picomole per
microliter.
Fluorescent Dye Selection
There are currently nine fluorescent dyes that are hard-coded for use with the MicroArray module (see table below). Users
can also enter & save fluorescent dyes not coded within the ND-1000 software using the ‘Dye/Chromophore Editor’ button
found in the main menu. Dyes can be selected using the scroll arrows or by highlighting the Dye 1 or Dye 2 box. The
respective absorbance wavelength, extinction coefficient, and 260nm and 280nm % corrections will be automatically
utilized for measurement and concentration calculation. The default settings from NanoDrop remain Dye 1 set to Cy3 and
Dye 2 set to Cy5. In addition to the fluorescent dyes available from the drop-down menu, an option entitled ‘None’ is also
available. Selecting ‘None’ disables the respective calculations & numeric displays corresponding to that dye.
Note: Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes.
Sample Volume Requirements
Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when
measuring aqueous nucleic acid samples containing incorporated fluorescent dyes. However, if you are unsure about the
surface tension properties of your sample or your pipettor accuracy, a 1.5-2 ul sample is recommended to ensure that the
liquid sample column is formed and the light path is completely covered by sample.
Measurement Concentration Range
The NanoDrop® ND-1000 Spectrophotometer will accurately measure fluorescent-dye and nucleic acid concentrations up
to 100 pmols/ul (Cy3) and 750 ng/ul (DNA) respectively without dilution. A table of sample concentration ranges is listed
below.
Sample
Type Detection Limit (pmol/ul) Approx. Upper Limit (pmol/ul)
Typical Reproducibility
(minimum 5 replicates)
(SD= pmol/ul; CV= %)
Cy3, Cy3.5, Alexa Fluor 555
and Alexa Fluor 660 0.20 100
sample range 0.20-4.0 pmol/ul:
±0.20 pmol/ul
sample range >4.0 pmol/ul: ±2%
Cy5, Cy5.5 and Alexa Fluor
647 0.12 60
sample range 0.12-2.4 pmol/ul:
±0.12 pmol/ul
sample range >2.4 pmol/ul: ±2%
Alexa Fluor 488 and Alexa
Fluor 594 0.40 215
sample range 0.40-8.0 pmol/ul:
±0.40 pmol/ul
sample range >8.0 pmol/ul: ±2%
Alexa Fluor 546 0.30 145 sample range 0.30-6.0 pmol/ul:
±0.30 pmol/ul
sample range >6.0 pmol/ul: ±2%
6-1

Section 6- MicroArray
Baseline Calculation & Normalization
The software normalizes the visual spectrum display for all readings at 750nm and will automatically calculate a baseline
between 400 and 750 nm for dye concentration calculations. The green vertical line on the screen represents the peak
wavelength position for Dye 1, and the red vertical line represents the peak wavelength position for Dye 2.
Unique Screen Features
Max Absorbance: used to rescale the upper limit of the vertical axis.
Sample Type: used to select the (color-keyed) type of nucleic acid being measured. The user can select ‘DNA-50’ for
dsDNA, ‘RNA-40’ for RNA, ssDNA-33’ for single-stranded DNA, or ‘Other’ for other nucleic acids. The default is ssDNA-
33’. If ‘other’ is selected, the user can select an analysis constant between 15-150. When navigating amongst the three
(3) general sample types within the Micro Array module, the last value of the constant entered within the ‘Constant’
Sample Type will be retained. See “Concentration Calculation (Beer’s Law)” in the appendix for more details on this
calculation.
λand Abs Norm: the user selected wavelength (black cursor) and corresponding absorbance at the 1mm pathlength.
The wavelength can be selected by dragging the black cursor or using the up/down arrows in the wavelength box. Note:
The user-selected wavelength and absorbance at the 1 mm pathlength are not utilized in any calculations.
Dye 1 (or 2): user selected dye
Abs. Norm: normalized absorbance of selected Dye at the 1 mm pathlength.
pmol/ul: concentration based upon selected Dye’s extinction coefficient. See “Concentration Calculation (Beer’s
Law)” in the appendix for more details on this calculation.
ng/ul: concentration of nucleic acids in the sample calculated using the absorbance at 260 nm minus the absorbance at
340 nm (i.e. normalized at 340 nm) and the nucleic acid analysis constant. See “Concentration Calculation (Beer’s Law)”
in the appendix for more details on this calculation.
260/280: ratio of sample absorbance at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess
the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted
as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other
contaminants that absorb strongly at or near 280 nm. See “260/280 Ratio’” section of the Troubleshooting section for
more details on factors that can affect this ratio.
6-2