Table of Contents
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ND-1000 Spectrophotometer
V3.5
User's Manual
NanoDrop Technologies, Inc.
3411 Silverside Road
Bancroft Building
Wilmington, DE 19810 USA
Voice: 302-479-7707
Fax: 302-792-7155
Email: info@nanodrop.com
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
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Table of Contents
Summary of Contents for NanoDrop ND-1000
- Page 1 Voice: 302-479-7707 Fax: 302-792-7155 Email: info@nanodrop.com 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...
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Page 2: Table Of Contents
Table of Contents Overview ....................1-1 Instrument Description ................1-1 Operation ....................1-1 Applications....................1-1 Patents ..................... 1-1 Initial Set Up ................... 2-1 Computer Requirements ................2-1 Software Installation ................. 2-1 Software Upgrades................... 2-2 Registering Your Instrument..............2-2 General Operation .................. 3-1 The Sample Retention System.............. - Page 3 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..............
- Page 4 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...
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Page 5: Overview
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). -
Page 6: Initial Set Up
“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. -
Page 7: Software Upgrades
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. -
Page 8: General Operation
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. -
Page 9: Software Architecture And Features
(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. -
Page 10: User Preferences
• 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’. -
Page 11: Utilities And Diagnostics
• 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. - Page 12 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.
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Page 13: Dye/Chromophore Editor
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. -
Page 14: Common Module Functions
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. -
Page 15: Re-Blank (F2)
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). -
Page 16: Print Report (F5)
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. -
Page 17: Nucleic Acids
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. -
Page 18: Spectrum Normalization
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”... -
Page 19: Microarray
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... -
Page 20: Baseline Calculation & Normalization
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. -
Page 21: Uv-Vis
0.1mm path for easier visual comparison. Sample ID label will be stored with sample data in the file folder “C:\NanoDrop Data\User name\ HiAbs”. This data cannot be imported into the Data Viewer but can be opened with Excel type spreadsheets. - Page 22 Section 7- UV/Vis Normalize: This is a user-selectable feature in this module. If selected, the software will automatically normalize the spectrum based on the lowest absorbance value in the range 400-750 nm. This feature may be selected as the default option using the User Preferences module.
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Page 23: Protein A280
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. -
Page 24: Spectrum Normalization
Section 8- Protein A280 The sample type (color-keyed) can be selected by clicking on the preferred option or by scrolling through the selections using the up or down arrow keys located to the left of the sample type box. A description of each sample type is given below. -
Page 25: Spectrum Overlay Control
Section 8- Protein A280 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. -
Page 26: Proteins & Labels
There are currently nine fluorescent dyes that are hard-coded for use with the Proteins and Labels 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. -
Page 27: Unique Screen Features
Section 9- Proteins & Labels Approx. Typical Reproducibility Sample Detection Upper (minimum 5 replicates) Type Limit Limit (SD= mg/ml; CV= %) sample range 0.10-10 mg/ml: ± 0.10 mg/ml Purified BSA 0.10 mg/ml 20 mg/ml sample range >10mg/ml: ± 2% sample range 0.20-4.0 pmol/ul: ± 0.20 pmol/ul sample range >4.0 pmol/ul: ±... -
Page 28: Protein Bca
Additionally, use the respective standard (e.g., BSA) and dilutions that cover the analytical range (mg/ml) of interest. Note: Since the ND-1000 can measure higher protein concentrations, you may need to supply your own protein standards at higher concentrations than provided by the manufacturer. -
Page 29: Making Bca Measurements
® NanoDrop ND-1000 Spectrophotometer software, it is recommended that the user follow manufacturers’ guidelines and generate fresh standard curves for each assay. Additionally, a standard curve ‘set-up’ may be reloaded. This feature will recall the respective standard series used in a previously saved standard curve. Both single and multi-point standard curve generation is incorporated into the software. -
Page 30: Standard Curve Features
Section 10- Protein BCA Step 2: Measure Standards Up to 5 replicates of each of up to 7 standards can be measured. The software will not allow measurement of samples until a minimum of 1 standard and references – or 2 standards –... -
Page 31: Exiting The Bca Module
Section 10- Protein BCA Regular BCA Standard Curve: 0.2 – 8.0 mg/ml mini-BCA Standard Curve: 0.01 – 0.20 mg/ml Exiting the BCA Module It is recommended that you process all of the unknowns to be assayed before exiting the BCA software module. 10-4... -
Page 32: Protein Lowry
Additionally, use the respective standard (e.g. BSA) and dilutions that cover the analytical range (mg/ml) of interest. Note: Since the ND-1000 can measure higher protein concentrations, you may need to supply your own protein standards at higher concentrations than provided by the manufacturer. -
Page 33: Making Lowry Measurements
® the NanoDrop ND-1000 Spectrophotometer software, it is recommended that the user follow manufacturers’ guidelines and generate fresh standard curves for each assay. Additionally, a standard curve ‘set-up’ may be reloaded. This feature will recall the respective standard series used in a previously saved standard curve. Both single and multi-point standard curve generation is incorporated into the software. -
Page 34: Standard Curve Features
Section 11-Protein Lowry Step 2: Measure Standards Up to 5 replicates of each of up to 7 standards can be measured. The software will not allow measurement of samples until a minimum of 1 standard and references – or 2 standards –... -
Page 35: Exiting The Lowry Module
Section 11-Protein Lowry Modified Lowry Standard Curve: 0.2 – 4.0 mg/ml Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module. 11-4... -
Page 36: Protein Bradford
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. -
Page 37: Unique Screen Features
Follow the manufacturer’s recommendation using standard (BSA) dilutions that cover the analytical (ug/ml) range of interest. Note: Since the ND-1000 Spectrophotometer can measure higher protein concentrations, you may need to supply your own protein standards at higher concentrations than provided by the manufacturer. -
Page 38: Standard Curve Features
Section 12- Protein Bradford Step 2: Measure Standards Up to 5 replicates of each of up to 7 standards can be measured. The software will not allow measurement of samples until a minimum of 1 standard and references – or 2 standards –... -
Page 39: Exiting The Bradford Module
Section 12- Protein Bradford Regular Bradford curve covers 200-8000 ug/ml. Note the linear range is 100-1000 ug/ml A mini-Bradford assay covers an approximate range of 15- 100 ug/ml. Exiting the Bradford Module It is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module. 12-4... -
Page 40: Cell Cultures
Cell Suspension Concentrations Due to its shorter pathlength, the ND-1000 can measure absorbencies that are 10-fold higher than those measurable on a standard cuvette spectrophotometer. This makes it possible to directly monitor concentrated cell suspensions. Since the entire spectrum is displayed, diluted samples exhibiting very low ‘Absorbance’ at 600 nm can be monitored at lower wavelengths, for example 280 nm. -
Page 41: Sample Homogeneity
Section 13- Cell Cultures Sample Homogeneity The user must be sure to homogeneously suspend the cells when sampling for “absorbance” measurements and read the sample immediately to avoid significant cell settling. Vigorous mixing may be required particularly when measuring concentrated samples 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 –... -
Page 42: Archived Data And Data Viewer
If the value is ‘Reblank’, it is the re-analysis of the previous measurement with a new blank. Data Storage Hierarchy The hierarchy for archive files is as follows: C:\NanoDrop Data User name Application Module (BCA Protein, Lowry, Bradford, Cell Culture, MicroArray, Nucleic Acid, Protein A-280, Proteins &... -
Page 43: Data Viewer
Show Report function found within each method module. It may also be accessed from the Main Menu page. An ND-1000 Spectrophotometer does not need to be connected to the PC to use the Data Viewer module. Data Viewer Features The Data Viewer is composed of two or three pages in a tabular form consisting of Plots, Reports and Standard Curves (where utilized). - Page 44 Archive File Converter: ND-1000 data generated with earlier versions of NanoDrop software and not stored in c:\nanodrop data will need to be copied and converted to 3.2 version compatible .ndj files. Refer to the Archive File Converter section below for more details. Note: The original archive files will not be altered during this process.
- Page 45 Section 14- Archived Data and Data Viewer Plots/Sets: Users may select the maximum number of individual plots (up to 20) graphed per page. Since a report can hold data for many samples and a graph page is limited to 20 plots, additional sample spectra are displayed on new pages.
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Page 46: Archive File Converter
All archive files located in the c:\Nanodrop Data folder generated with earlier versions of NanoDrop software will automatically be copied and converted to version-compatible .ndj files upon first use of the software. Archive files generated with earlier versions of software and not stored in the folder c:\NanoDrop Data will need to be converted 14-5... -
Page 47: Opening Archived Data With Spreadsheet Programs
Section 14- Archived Data and Data Viewer manually before reviewing with the Data Viewer. The File Converter can be accessed from the Import Data page and the conversion can be done one file at a time or by converting an entire file folder. Note: The original archive files are not altered during this process. -
Page 48: Calibration Check
Procedure Ensure the measurement pedestals are clean and that a 1ul water sample “beads” up on the lower pedestal. 1.) Open the ND-1000 Calibration Check Software and follow the prompts in the Customer Guidance text box of the software. 2.) Enter the Target Absorbance found on the CF-1 vial as directed in the image below, Typically the target absorbance is 0.734;... - Page 49 Section 15- Calibration Check NOTE: The CF-1 Calibration Fluid is supplied in a single use vial. The CF-1 must be used within one hour of opening the vial. Exposure to the environment or transferring of the fluid to another container may cause a significant change in concentration.
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Page 50: Troubleshooting
Other Windows Operating Systems 4. Try the NanoDrop software, if it works properly you are finished. If it does not operate properly, go to step 5. 5. Shutdown the NanoDrop software and open the USBView utility to confirm proper USB communication: Start... -
Page 51: Connection Error
Section 16- Troubleshooting Connection Error This error occurs whenever the USB connection is disrupted while operating a software module. In most cases, selecting ‘Retry’ will reconnect properly. Some possible causes and solutions are listed below: Power management scheme on the PC: If your PC is automatically going into standby or hibernate mode, the USB communication will be lost whenever it occurs and ‘Retry’... -
Page 52: Saturated Detector
It is not necessary to close the software completely as each module is re-initialized when it is opened. If the error persists, contact NanoDrop Technologies or your local distributor Liquid Column Breakage... - Page 53 If the warning persists and the user visually confirms that the liquid column is forming, contact NanoDrop Technologies or your local distributor.
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Page 54: Other Software Error Messages
This error code is most likely to occur if the Windows account that is currently logged into Windows does not have read and write access to the folder c:\nanodrop data or one of its subfolders. See your PC administrator to make sure that all users of the NanoDrop software operate with a Windows account that has the appropriate access. -
Page 55: Sample Accuracy And Reproducibility
Heat DNA samples to 55 °C and vortex before measurement Due to the small volumes required by the ND-1000, it is extremely important to ensure that the sample being measured is homogeneous. Field experience has shown that samples containing large molecules such as genomic or lambda DNA are particularly susceptible to this phenomenon. -
Page 56: 260/280 Ratio
Confirm instrument accuracy and reproducibility with CF-1 This is a potassium dichromate calibration standard available from NanoDrop Technologies and its distributors. It is a good practice to check the instrument’s performance every six months with a fresh vial of CF-1. -
Page 57: Technical Service
A spectrum that is very “un-smooth” or “ragged” can be caused by insufficient light intensity reaching the spectrometer. If you suspect that this is occurring, refer to the “Technical Service” section for instructions on how to contact NanoDrop Technologies and what information must be sent to allow for diagnosing the problem. -
Page 58: Maintenance And Warranty
All spectrophotometers and accessories manufactured by NanoDrop Technologies are warranted against manufacturing defects in parts and labor for a period of one year. Preventive Maintenance as well as additional one, two, and three year warranty extensions are available. Plan descriptions can be found at the following link: www.nanodrop.com. 17-1... -
Page 59: Appendices
(M). Fluorescent Dyes (Microarray Measurement) The NanoDrop software uses the general form of the Beer-Lambert equation to calculate fluorescent dye concentrations in the Microarray module. The table of extinction coefficients for each dye is below: Extinction... - Page 60 • RNA: 40 ng-cm/ul ® For the NanoDrop ND-1000 Spectrophotometer, path lengths of 1.0 mm and 0.2 mm are used compared to a standard ® spectrophotometer using a 10.0 mm path. Thus, the NanoDrop ND-1000 Spectrophotometer is capable of measuring samples that are 50 times more concentrated than can be measured in a standard spectrophotometer.
- Page 61 8. Click on the ‘Device Settings’ tab and ensure 30256 Shipping Label is selected as the ‘Default’ label. It is best to confirm that the shipping label selection has been recognized by checking the set-up from within the NanoDrop software.