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Related Manuals for Thermo Scientific NanoDrop 1000 V3.3
Summary of Contents for Thermo Scientific NanoDrop 1000 V3.3
- Page 1 ND-1000 Spectrophotometer V3.3 User’s Manual...
- Page 2 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 ©...
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Page 3: 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................... 2-1 Registering Your Instrument ..............2-2 General Operation .................. 3-1 The Sample Retention System .............. - Page 4 Measurement Concentration Range............9-1 Unique Screen Features................9-1 Baseline Type................... 9-2 10. Protein BCA ..................10-1 Sample Volume Requirements ............... 10-1 Special Cleaning Requirements for Proteins .......... 10-1 Measurement Concentration Range............10-1 BCA Kits, Protocols, and Sample Preparation........10-1 Unique Screen Features................. 10-1 Making BCA Measurements ..............
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Page 5: Overview
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. -
Page 6: Initial Set Up
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. No Mac versions of the software are currently available. • Microsoft Windows XP or 2000 operating system. Windows Vista has also been tested successfully with NanoDrop software. -
Page 7: Registering Your Instrument
Section 2- Initial Set Up 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. ®... -
Page 8: General Operation
Section 4-Common Module Functions 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. -
Page 9: Software Architecture And Features
Section 4-Common Module Functions 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: •... -
Page 10: User Preferences
Section 4-Common Module Functions 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 •... - Page 11 Section 4-Common Module Functions 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 below). 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.
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Page 12: Dye/Chromophore Editor
Section 4-Common Module Functions 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. -
Page 13: 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. When this message disappears, the instrument will be ready for use. -
Page 14: Re-Blank (F2)
Section 4-Common Module Functions 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. -
Page 15: Show Report (F7)
Section 4-Common Module Functions 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. -
Page 16: 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 17: Spectrum Normalization
Section 5- Nucleic Acids Show Report: formatted for 200 samples although the buffer size can be increased to accommodate thousands of samples. 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. -
Page 18: Microarray
Section 6- MicroArray 6. MicroArray The capability to pre-select viable fluorescent-tagged hybridization probes for gene expression in microarrays 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). -
Page 19: Baseline Calculation & Normalization
Section 6- MicroArray 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. -
Page 20: Uv-Vis
Section 7- UV-VIS 7. UV-VIS ® The ‘UV/VIS Absorbance’ module allows the NanoDrop ND-1000 Spectrophotometer to function as a conventional spectrophotometer. Sample absorbance is displayed on the screen from 220 nm to 750 nm and cursors permit the measurement of individual peaks. Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring most aqueous samples. -
Page 21: Protein A280
Section 8- Protein A280 8. Protein A280 Proteins, unlike nucleic acids, can exhibit considerable diversity. The A280 method is applicable to purified proteins exhibiting absorbance at 280nm. It does not require generation of a standard curve and is ready for quantitation of protein samples at startup. This module displays the UV spectrum, measures the protein’s absorbance at 280 nm (A280) and calculates the concentration (mg/ml). -
Page 22: Spectrum Normalization
Section 8- Protein A280 IgG reference. Unknown (sample) protein concentrations are calculated using the mass extinction coefficient of 13.7 at 280 nm for a 1% (10 mg/ml) IgG solution. Lysozyme reference. Unknown (sample) protein concentrations are calculated using the mass extinction coefficient of 26.4 at 280 nm for a 1% (10 mg/ml) Lysozyme solution. -
Page 23: Proteins & Labels
Section 9- Proteins & Labels 9. Proteins & Labels This software module can be used to determine protein concentration (A280nm) as well as fluorescent dye concentration (protein array conjugates), or to measure the purity of metalloproteins (such as hemoglobin) using wavelength ratios. Fluorescent Dye Selection There are currently nine fluorescent dyes that are hard-coded for use with the Proteins and Labels module (see table below). -
Page 24: Baseline Type
Section 9- Proteins & Labels Max Absorbance: used to rescale the upper limit of the vertical axis. Sample Type: The same six sample types (options) listed under Protein A280 (Section 8) are available for purified Proteins & Labels analysis and concentration measurement. All of the options can be viewed by clicking the mouse while it is positioned within the sample type box. -
Page 25: Protein Bca
Section 10- Protein BCA 10. Protein BCA The BCA (Bicinchoninic Acid) Protein Assay is an alternative method for determining protein concentration. It is often used for more dilute protein solutions and/or in the presence of components that also have significant UV (280 nm) absorbance. Unlike the Protein A280 method, the BCA Assay requires a standard curve to be generated each time it is run, before unknown proteins can be measured. -
Page 26: Making Bca Measurements
Section 10- Protein BCA mg/ml: the concentration of the sample (unknown). Show Report: formatted for 200 samples although the buffer size can be increased to accommodate thousands of samples. Making BCA Measurements ® A standard curve is required every time the BCA assay is run. Although curves can be saved and reloaded in the NanoDrop ND-1000 Spectrophotometer software, it is recommended that the user follow manufacturers’... -
Page 27: 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 exciting the BCA software module. 10-3... -
Page 28: Protein Lowry
Section 11-Protein Lowry 11. Protein Lowry The Modified Lowry Protein Assay is an alternative method for determining protein concentration based on the widely used and cited Lowry procedure for protein quantitation. Like the BCA and Bradford Assays, the Modified Lowry Assay requires standard curve generation each time it is run, before unknown proteins can be measured. -
Page 29: Making Lowry Measurements
Section 11-Protein Lowry Show Report: formatted for 200 samples although the buffer size can be increased to accommodate thousands of samples. Making Lowry Measurements ® A standard curve is required every time the Modified Lowry assay is run. Although curves can be saved and reloaded in the NanoDrop ND-1000 Spectrophotometer software, it is recommended that the user follow manufacturers’... -
Page 30: 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 exciting the Lowry software module. 11-3... -
Page 31: Protein Bradford
Section 12- Protein Bradford 12. Protein Bradford The Bradford Assay is a second alternative method commonly utilized for determining protein concentration. It is often used for more dilute protein solutions where lower detection sensitivity is needed and/or in the presence of components that also have significant UV (280 nm) absorbance. -
Page 32: Making Bradford Protein Measurements
Section 12- Protein Bradford Reset Window (F11): clears all replicates of all standards. Reset This Std (F12): clears all replicates of the selected standard. Absorbance at 595nm: protein-dye complex’s absorbance at 595 nm at the 1mm pathlength. Cursor λ and Absorbance: this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance. The cursor wavelength can be set by the user. -
Page 33: Standard Curve Features
Section 12- Protein Bradford Standard Curve Features Standard curves can be saved and reloaded for reference use by using the ‘Standard Curve’ pull down menu and choosing save or reload functions. Selecting the ‘View Standard Curve’ button allows the user to review the standard curve at any time. The Delete Point button allows the user to delete points by first selecting the data point to be deleted in the table and then choosing the ‘Delete Point’... -
Page 34: Cell Cultures
Section 13- Cell Cultures 13. Cell Cultures Using an absorbance spectrophotometer to monitor light scattered by non-absorbing suspended cells is common practice in life science laboratories. Such applications, more than any other, accentuate the differences amongst the optical systems of the numerous spectrophotometer designs. -
Page 35: Decontamination Of Measurement Pedestals
Section 13- Cell Cultures Decontamination of Measurement Pedestals If decontamination is necessary, a 5.25% solution of sodium hypochlorite (bleach – freshly prepared) or other decontaminating solutions can be used to ensure that no biologically active material is present on the measurement pedestals. The metal fiber optic fittings are made from 303 stainless steel and are very resistant to most common laboratory solvents (see “Solvent Compatibility”... -
Page 36: Archived Data And Data Viewer
Section 14-Archived Data and Data Viewer 14. Archived Data and Data Viewer Sample data from all application modules is automatically stored in archive files and can be opened by either the integrated Data Viewer software program or spreadsheet programs such as MS Excel. Archive File Creation Every time an application module is started, an application-specific archive file is created for the user that is logged in. -
Page 37: Data Viewer
Section 14-Archived Data and Data Viewer Data Viewer Data Viewer is a versatile data reporting software program incorporated into the NanoDrop software that offers the user the ability to customize report structures, import stored data and re-plot data generated from NanoDrop instruments. Using the Data Viewer is the most expedient method to review data. - Page 38 Section 14-Archived Data and Data Viewer >>> or <<<: Used to move the highlighted sample choices to or from the Selected Samples box. Note: The software defaults to a buffer size of 200 samples. Search Function allows the user to locate specific data by searching through sample ID names. Sample Information and Spectrum: Are populated with the information associated with the most recently highlighted sample Import and Return: Uses selected sample data to populate Plots and Reports windows.
- Page 39 Section 14-Archived Data and Data Viewer Reports The Reports page displays the data for selected samples in a table format. The user may modify column configurations for each method type and save multiple customized formats. Some key options useful for the Reports page are accessible through the Report tool bar drop down. Choosing the ‘Configure Report’...
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Page 40: Archive File Converter
Section 14-Archived Data and Data Viewer Using the Full Report option will allow the user to use the Data Viewer to reload the report at a later date. The saved report may be recalled using the pull down Load Report. If using the Load Report feature- the report will be displayed with the default column configuration. -
Page 41: Opening Archived Data With Spreadsheet Programs
Section 14-Archived Data and Data Viewer Opening Archived Data with Spreadsheet Programs The archived files are in tab-delimited format and can be opened in Microsoft Excel or an equivalent spreadsheet program. 14-6... -
Page 42: Troubleshooting
Section 15- Troubleshooting 15. Troubleshooting Error USB2000 This error might appear upon software startup and usually indicates that the USB cable is not properly connected or the software is not loaded properly. To troubleshoot, do the following: 1. Confirm that the USB cable is connected to both the PC and the instrument. 2. -
Page 43: Connection Error
Section 15- 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 44: Saturated Detector
Section 15- Troubleshooting • With the sampling arm down, select OK to initialize the spectrometer and then select ‘Intensity Check’. You should see a red and black spectrum and a bias value greater than 65 as shown below. This indicates that the USB communication is normal, the power supply is operational and the flashlamp is functioning. -
Page 45: Other Software Error Messages
Section 15- Troubleshooting Proteins and solutions containing surfactants are known to “un-condition” the measurement pedestal surfaces so that the liquid column does not form. “Re-condition the pedestal as follows: • Apply 5-10 ul of ~0.5M of HCl to the bottom pedestal, lower the upper pedestal into the solution and let it sit for approximately 2 to 3 minutes. -
Page 46: Sample Accuracy And Reproducibility
Section 15- Troubleshooting This error indicates that there is insufficient light getting through to make good absorbance measurements. Check that the sampling arm is in the down position and the power is connected. Error 9003 This error indicates that the monitor resolution is below the 1024x768 required. Check the computer settings. Be sure that the start menu tool bar is set to the bottom and not along the side. -
Page 47: 260/280 Ratio
Section 15- Troubleshooting Analyze a fresh replicate of the blanking solution as though it were a sample by selecting ‘Measure’ (F1). The result should be a spectrum that varies no more than 0.050 A (10mm absorbance equivalent). Wipe the blank from both measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum is within 0.005 A (1mm path). -
Page 48: Technical Service
Section 15- Troubleshooting Detector saturation- nucleic acid Detector saturation- Bradford measurement measurement 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 49: Maintenance And Warranty
Section 16- Maintenance and Warranty 16. Maintenance and Warranty Cleaning ® The primary maintenance requirement of the NanoDrop ND-1000 Spectrophotometer is keeping the measurement pedestal surfaces clean. Upon completion of each sample measurement, wipe the sample from the upper and lower pedestals to prevent sample carryover and avoid residue buildup. -
Page 50: Appendices
Section 17- Appendices 17. Appendices Instrument Specifications • Sample Size: 1microliter • Path Length: 1 mm (with auto-ranging to 0.2 mm) • Light Source: Xenon flash lamp • Detector Type: 2048-element linear silicon CCD array • Wavelength Range: 220-750 nm •... -
Page 51: Solvent Compatibility
Section 17- Appendices For nucleic acid quantification, the Beer-Lambert equation is modified to use an extinction coefficient with units of ng-cm/ml. Using this extinction coefficient gives a manipulated equation: c = (A * e)/b Where c is the nucleic acid concentration in ng/microliter, A is the absorbance in AU, e is the wavelength-dependent extinction coefficient in ng-cm/microliter and b is the path length in cm.