NanoDrop 2000/2000c Spectrophotometer V1.
The information in this publication is provided for reference only. All information contained in this publication is believed to be correct and complete. Thermo Fisher Scientific shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing, performance or use of this material. All product specifications, as well as the information contained in this publication, are subject to change without notice.
Table of Contents 1. INTRODUCTION ................................................................................................................................................................ 1-1 INSTRUMENT DESCRIPTION ................................................................................................................................................... 1-1 INSTRUMENT SPECIFICATIONS ................................................................................................................
Unique Screen Features .................................................................................................................................................. 3-30 Making Lowry Assay Measurements.............................................................................................................................. 3-32 PROTEIN BRADFORD ...........................................................................................................................................................
Section 1-Introduction 1. Introduction Instrument Description Thermo Scientific NanoDrop™ 2000/2000c Spectrophotometers measure 0.5- 2 ul samples with high accuracy and reproducibility. The NanoDrop 2000c model offers the convenience of both the NanoDrop patented sample retention technology and a traditional cuvette for sample measurements. The sample retention system employs surface tension to hold the sample in place between two optical fibers.
Section 1-Introduction Sample Retention Pedestal Measurements A 1 - 2 µL sample is pipetted onto a measurement pedestal. A smaller, 0.5 µL volume sample, may be used for concentrated nucleic acid and protein A280 samples. A fiber optic cable (the receiving fiber) is embedded within this pedestal. 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 ends of the two fibers.
Section 1-Introduction Cuvette Measurements The NanoDrop 2000c will accept 10 mm cuvettes up to 48 mm tall. When measuring samples using micro, semimicro, or ultra-micro cuvettes, we recommend using masked cuvettes. Masked cuvettes ensure that all light hitting the detector has passed through the sample. Unmasked cuvettes can allow light to hit the detector that has not passed through the sample.
Section 1-Introduction Blanking and Absorbance Calculations When a NanoDrop 2000/2000c spectrophotometer is blanked, a spectrum is taken of the reference solution (blank) and stored in memory as an array of light intensities by wavelength. When a measurement of a sample is taken, the intensity of light that was transmitted through the sample is recorded.
Section 1-Introduction Fluorescent Dyes The software uses the general form of the Beer-Lambert equation to calculate fluorescent dye concentrations in the Micro Array and Proteins & Labels applications. The user can choose to enter new dye types using the Dye/Chrom. Editor or use one of the predefined dyes.
Section 2-Software 2. Software Computer Requirements • • • • • • Microsoft Windows XP or Vista (32 bit) operating system 1.5GHz or higher processor CD ROM drive 1GB or more of RAM (2GB if running Vista) 40 MB of free hard disk space Available USB port (the instrument uses a USB cable to connect to the computer) Software Installation 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.
Section 2-Software Cable Connections To make measurements with the instrument, connect the USB cable to the instrument and the PC, plug in the 12V power supply and connect to the power input at the back of the instrument. The power supply can remain plugged into the NanoDrop 2000/2000c while the instrument is not in use. When the unit is in this “standby” mode, power consumption is ~5 W and the flash lamp is not energized.
Section 2-Software • Dye/ Chrom. Editor (Micro Array and Protein & Labels only) - enables the user to enter and edit new dyes in addition to predefined dyes. Refer to “Dye/Chrom. Editor” within “Micro Array” and “Protein & Labels” for additional details. • Editor Options (within Method Editor) – defines the available options for editing methods. Refer to “Method Editor” for additional information.
Section 2-Software - E-mail current workbook - automatically attaches the current workbook to a new email message. When sending data to NanoDrop products technical support, please use the My Data task bar to locate the appropriate autosave data file. Open the file with the NanoDrop 2000/2000c software and send to Technical Support. Note: Although all Report fields may not be displayed when opening an autosave file, all fields are recoverable by the Technical Support team.
Section 2-Software Right Pane The right pane displays the main menu which includes the following: • Group drop-down box - selects the preferred main menu display and associated applications. The default selection is the Classic NanoDrop group of applications. • Predefined application buttons - and additional main menu selections. • User-Defined list - empty until user defined methods or kinetics methods are created.
Section 2-Software • Sample labels (UV-Vis only) - selects whether absorbance values for specific wavelengths are displayed on the graph for the most recent sample measurement. • Sample legend - determines whether or not the samples IDs are displayed on the top left hand side of the graph.
Section 2-Software Data and Account Management My Data Sample measurement data is recorded in workbooks that can be filed in a user-specified location. The left pane My Data task bar is used to access saved workbooks. Use the navigator pane to find the folder where the workbook of interest is stored. Two icons are available at the top of the left pane once the My Data task bar is selected. Selecting New opens a new workbook for the highlighted application in the right pane.
Section 2-Software - Report, Excel XML Spreadsheet (*.xml) - saves a report in a format which can be opened in Excel. Only the columns displayed in the report will be saved using this feature. It is important to configure the report to display the information desired prior to exporting. - Report, Tab Separated Values (*.tsv) - saves a report in a format which can be opened in Notepad or Excel. Only the columns displayed in the report will be saved using this feature.
Section 2-Software Reprocess The Reprocess feature is available in the top left pane of the reports page for some applications. This feature recalculates a sample concentration based upon the selection of alternative baseline correction wavelengths, concentration units and/or sample types. This feature is not used to rename samples. A Reprocess field is available as a report configuration column option. Note: Reprocessed sample data will appear in the current report as a new data entry.
Section 2-Software • Report Master Page - defines the layout and appearance of headers and footers for printed pages. Using this tab, parameters such as whether or not to include an organization name, a logo, the date and/or the time in a header are selected. Footer options include text additions and page numbers. Selections will be applied to printed reports for all applications and user-defined methods. The Page Setup icon brings up the window used for determining paper size and printer margins.
Section 2-Software The left side of the Accounts page displays a list of access control categories. Each of these categories includes software features to which specified users, or groups of users, can be granted access. The two categories of access include: - Allow access to: - Options - enables users or groups to have access to: Applications, Report Master Page, Preferences, and Accounts.
Section 2-Software Drop downs and list boxes: • List names from - displays a list of available domains from which to choose. • Organization - displays a list of organizations within selected domain. (This list is not available if a local domain is selected.) • Allow access to - the list of users and groups selected to be granted access to specified software features as described above.
Section 3- Applications 3. Applications Overview UV-VIS spectrophotometry is easy for micro-volume samples using NanoDrop 2000/2000c spectrophotometers.
Section 3- Applications A Quick Start Guide that includes a blanking procedure can be accessed from “Appendices” of the NanoDrop 2000 User Manual PDF. It is recommended that this guide be printed and posted near the instrument as a reference. Measurement Ranges Sample Type Nucleic Acids Lower Detection Limit 2 ng/µL (pedestal) 0.4 ng/µL (cuvette) Microarray Protein A280 Approximate Upper Detection Limit 2 - 100 ng/uL: + 2 ng/uL < 15,000 ng/ µL (dsDNA) 2 ng/ µL (pedestal) 0.4 ng/ µL (cuvette) 0.
Section 3- Applications Nucleic Acid Overview Nucleic acid samples can be easily checked for concentration and quality using the NanoDrop 2000/2000c spectrophotometer. To measure nucleic acid samples select the Nucleic Acid application from the home page. Nucleic Acid Calculations For nucleic acid quantification, the Beer-Lambert equation is modified to use a factor with units of ng-cm/microliter.
Section 3- Applications Unique Screen Features The right pane displays features specific to the Nucleic Acid application. Task bars in the left pane not described below are described in “Software Overview.” The spectral display shows data for the current sample normalized to a 10 mm path for all measurements including measurements made with any cuvette pathlength. The following features are to the right of the spectral display: • Sample ID - field into which a sample ID is entered.
Section 3- Applications • 260/230 - ratio of absorbance at 260 nm and 230 nm. This is a secondary measure of nucleic acid purity. The 260/230 values for a “pure” nucleic acid are often higher than the respective 260/280 values and are commonly in the range of 1.8-2.2. If the ratio is appreciably lower, this may indicate the presence of copurified contaminants. • Baseline correction - if selected, the default wavelength for the bichromatic normalization is 340 nm.
Section 3- Applications After the measurement: - Simply wipe the upper and lower pedestals using a dry laboratory wipe and the instrument is ready to measure the next sample. - When using the cuvette option, remove the cuvette, rinse thoroughly and dry between samples. Oligo Calc Oligo Calc is used to calculate molecular weights, extinction coefficients, concentration factors and melting points for specific nucleic acid sequences.
Section 3- Applications To calculate the melting point of a DNA sequence: 1. Enter the base sequence as described above. If a base sequence has already been entered in the Oligo Calc Tab, then the base sequence box in the Melting Points tab will be auto-populated with that sequence. 2. Enter the appropriate values in the each of the boxes as described below: - Oligo Molarity- enters the oligo molarity of the sample.
Section 3- Applications Micro Array Overview The capability to pre-select viable fluorescently-tagged hybridization probes for use on micro arrays can eliminate potentially flawed samples and improve research effectiveness. The NanoDrop 2000/2000c measures the absorbance of the fluorescent dye, allowing detection at dye concentrations as low as 0.2 picomole per microliter. The software automatically utilizes the optimal pathlength to measure the absorbance of each sample.
Section 3- Applications The spectral display shows data for the current sample normalized to a 1 mm path if measured using the pedestal mode or the pathlength selected if using the cuvette mode (model 2000c only). The pathlength information is indicated on the Y axis. The following features are to the right of the spectral display: • Sample ID- field into which a sample ID is entered. The appropriate sample ID should be entered prior to each measurement.
Section 3- Applications 4. Use the drop-down lists in the Dye 1 or Dye 2 box to select the appropriate dye (or dyes). The default for Dye 1 is Cy3 and the default for Dye 2 is Cy5. If the nucleic acid has been labeled with only one dye, choose None as the dye type for Dye 2. 5. A default wavelength of 340 nm is automatically used for a bichromatic normalization for the nucleic acid component of the sample.
Section 3- Applications To use the Oligo Calc: 1. Use one of the following options to enter in a base sequence: - The buttons below the Base Sequence display. - The keyboard. (Only the A, C, G, T, and U keys will work to enter bases.) - Copy and paste a base sequence into the display from another application. (Only the letters A, C, G, T, and U can be pasted into the Base Sequence display.) - To clear the Base Sequence display, click Clear to the right of the display.
Section 3- Applications Melting Point analysis result fields include: • Salt-Adjusted - calculates the melting point of the base sequence without accounting for the effect of interaction between neighboring bases. • Nearest-Neighbor - displays the melting point of the base sequence when the effect of interaction between neighboring bases is taken into account.
Section 3- Applications UV-Vis Overview The UV-Vis application allows the NanoDrop 2000/2000c to function as a conventional spectrophotometer. Sample absorbance is displayed on the screen from 190 nm to 840 nm. Up to 40 wavelengths can be designated for absorbance monitoring and inclusion in the report. Measurement Concentration Ranges The NanoDrop 2000/2000c can measure samples with 10 mm pathlength absorbance equivalent of 300 A when using the auto pathlength feature.
Section 3- Applications - The shorter pathlength is advantageous for highly concentrated samples. Although this is an automatic function in other applications, the user does have the option of using the auto pathlength feature or restricting the pedestal to a 1 mm pathlength in the UV-Vis application. In either case, the absorbance data will be displayed as 1 mm equivalent absorbance values.
Section 3- Applications recommended that cuvettes be removed from the instrument prior to making a pedestal measurement to ensure that the pedestal arm can move to the proper starting position. 7. Enter a sample ID in the appropriate field, load the first sample as described for the blank above and click Measure. Note: A fresh aliquot of sample should be used for each measurement.
Section 3- Applications Protein A280 Overview Proteins, unlike nucleic acids, can exhibit considerable diversity. The Protein A280 application is applicable to purified proteins that contain Trp, Tyr residues or Cys-Cys disulphide bonds and exhibit absorbance at 280 nm. This method does not require generation of a standard curve and is ready for protein sample quantitation at software startup.
Section 3- Applications Unique Screen Features The right pane displays features specific to the Protein A280 application. Task bars in the left pane not described below are described in “Software Overview.” The spectral display shows data for the current sample normalized to a 10 mm path for all measurements including measurements made with any cuvette pathlength (2000c only). The following features are to the right of the spectral display: • Sample ID - field into which a sample ID is entered.
Section 3- Applications • Ext. Coeff, E1% L/gm-cm - appears when Other Protein (E1%) is selected. The appropriate extinction coefficient should be entered prior to making a measurement. • ε/1000 and M.W. (KDa) - appears when Other protein (E & MW) is selected. The appropriate e/1000 and M.W. (kDa) should be entered prior to making a measurement. • Conc. - concentration based on absorbance at 280 nm and the selected extinction coefficient.
Section 3- Applications 8. Enter a sample ID in the appropriate field, load the first sample as described for the blank above and click Measure. Note: A fresh aliquot of sample should be used for each measurement. After the measurement: - Simply wipe the upper and lower pedestals using a dry laboratory wipe and the instrument is ready to measure the next sample. - When using the cuvette option, remove the cuvette, rinse thoroughly and dry between samples.
Section 3- Applications Proteins & Labels Overview The Proteins & Labels application can be used to determine protein concentration (A280 nm) as well as fluorescent dye concentration (protein array conjugates). It can also be used to measure the purity of metalloproteins (such as hemoglobin) using wavelength ratios.
Section 3- Applications Unique Screen Features The right pane displays features specific to the Protein & Labels application. Task bars in the left pane not described below are described in “Software Overview.” The spectral display shows data for the current sample normalized to a 10 mm path for all measurements including measurements made with any cuvette pathlength (model 2000c only). The following features are to the right of the spectral display: • Sample ID - field into which sample ID is entered.
Section 3- Applications • uM - is the concentration based upon the respective dye’s extinction coefficient. Concentration units may be selected from the adjacent drop-down list box. A280 - displays the absorbance at 280 nm for the protein sample measured. The displayed absorbance value is normalized to a 10 mm path. Note: The displayed A280 value is the difference in absorbance of the sample at 280 nm compared to the absorbance of the sample at 750 nm.
Section 3- Applications - Cuvette Option (Model 2000c only): Insert the cuvette noting the direction of the light path indicated by the etched arrow. The optical beam (2 mm) is directed 8.5 mm above the bottom of the cuvette. Refer to the cuvette manufacturer for volume recommendations. Note: The arm must be down for all measurements, including those made with cuvettes.
Section 3- Applications Protein BCA Overview The BCA (Bicinchoninic Acid) assay is a colorimetric method for determining the total protein concentration in unpurified protein samples. It is often used for dilute protein solutions and/or proteins in the presence of components that have significant UV (280 nm) absorbance. Unlike the Protein A280 application, the Protein BCA application requires a standard curve be generated before sample protein concentrations can be measured.
Section 3- Applications Pedestal Reconditioning Solutions and reagents containing surfactants may “un-condition” the measurement pedestal surfaces so that the liquid column does not form. If this occurs, 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 PR-1 may be found on our website.
Section 3- Applications • Standards radio button - enables entry of standard names and concentrations into the standards table when selected. • Concentration mg/mL - concentration of current sample in mg/mL. • Absorbance at 562 nm - the Cu-BCA complex’s absorbance at 562 nm. BCA Standard Curves A standard curve is required for BCA assays.
Section 3- Applications Note: If selecting a previously saved workbook, all concentration calculations for newly measured samples will be based upon the standard absorbance values saved in the workbook. Each workbook will archive only one standard curve. Making BCA Assay Measurements • Refer to the manufacturers’ guidelines and recommendations for sample preparation.
Section 3- Applications Note: A fresh aliquot of sample should be used for each measurement. After the measurement: - Simply wipe the upper and lower pedestals using a dry laboratory wipe and the instrument is ready to measure the next sample. - When using the cuvette option, remove the cuvette, rinse thoroughly and dry between samples.
Section 3- Applications Protein Lowry Overview The Lowry assay is an alternative method for determining protein concentration based on the widely used and cited Lowry procedure for protein quantitation. Like the other colorimetric assays, the Lowry Assay requires a standard curve be generated before sample proteins can be measured. The Lowry procedure involves reaction of protein with cupric sulfate in alkaline solution, resulting in formation of tetradentate copper-protein complexes.
Section 3- Applications Unique Screen Features The right pane displays features specific to the Protein Lowry application. Task bars in the left pane not described below are described in “Software Overview.” The spectral display shows data for the current sample normalized to a 1 mm path if measured using the pedestal mode or the pathlength selected if using the cuvette option (model 2000c only). The pathlength information is indicated on the Y axis.
Section 3- Applications Lowry Standard Curves A standard curve is required for Lowry assays. Example Standard Curve: 5:1 reagent/sample volume. • A standard curve can be generated using a minimum of two points which may include two standards or a reference (Lowry reagent/assay buffer only – no protein) and one standard. • The multi-point curve capability allows for multiple replicates for up to seven standards. There is no set order in which standards must be run.
Section 3- Applications Making Lowry Assay Measurements • Refer to the manufacturers’ guidelines and recommendations for sample preparation. • The Zero Reference standard is a solution of the same buffer/dye reagent composition as the other standards and samples but without any protein added. • Prepare both standards and unknowns in the same manner. Use a diluent of the same pH and ionic strength for all blanks, standards and unknown samples.
Section 3- Applications After the measurement: - Simply wipe the upper and lower pedestals using a dry laboratory wipe and the instrument is ready to measure the next sample. - When using the cuvette option, remove the cuvette, rinse thoroughly and dry between samples.
Section 3- Applications Protein Bradford Overview The Bradford Assay is a commonly used method 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. Like the other colorimetric assays, the Bradford assay requires a standard curve be generated before sample proteins can be measured.
Section 3- Applications When using the cuvette option, it is essential that sufficient sample volume be used to ensure that the light path is passing through a representative portion of the sample. The optical beam (2 mm) is directed 8.5 mm above the bottom of the cuvette. Refer to the cuvette manufacturer for recommended volumes. Pedestal Reconditioning Solutions and reagents containing surfactants may “un-condition” the measurement pedestal surfaces so that the liquid column does not form.
Section 3- Applications • Samples radio button – Once the criteria have been met for a valid curve, the Sample ID field will become active when selected. The sample ID should be entered prior to making the measurement. The sample ID should be entered prior to making the measurement. • Standards radio button – enables entry of standard names and concentrations into the standards table when selected. • Concentration ug/ml - concentration of current sample in ug/ml.
Section 3- Applications • In order to establish a new standard curve, a new workbook must be created. Colorimetric applications will offer several options regarding the use of previous standard curves when appending sample data to previous workbooks. It is recommended that the user follow the protein assay manufacturer’s guidelines for generating standard curves before appending new data to a workbook.
Section 3- Applications 7. After all of the Standards have been measured, click on the Samples radio button. Enter a sample ID. Load 2 µL of sample when using the pedestal or insert the cuvette. Click Measure to initiate the measurement. It is not necessary to blank the instrument between the standard and the unknown sample measurements. Note: A fresh aliquot of sample should be used for each measurement.
Section 3- Applications Protein Pierce 660 nm Overview The Thermo Scientific Protein Pierce 660 nm assay reagent is a ready-to-use formulation that offers rapid, accurate and reproducible colorimetric detection of minute amounts of protein in solution. The reagent is ideal for measuring total protein concentration in samples containing reducing agents and/or detergents. Protein Pierce 660 nm Reagent and Protocols This protocol uses a 15:1 reagent/sample volume ratio.
Section 3- Applications Unique Screen Features The right pane displays features specific to the Protein Pierce 660 nm application. Task bars in the left pane not described below are described in “Software Overview.” The spectral display shows data for the current sample normalized to a 1 mm path if measured using the pedestal mode or the pathlength selected if using the cuvette option (model 2000c only). The pathlength information is indicated on the Y axis.
Section 3- Applications Protein Pierce 660 nm Assay Workbooks When the Protein Pierce 660 nm application is selected from the main menu, the following message will appear: Although standard curve absorbance values may be entered manually rather than measured, it is recommended that the user follow the manufacturer’s suggestions for generating new standard curves for each assay. In order to establish a new standard curve, a new workbook must be created.
Section 3- Applications Note: This is unlike the other colorimetric assays on the NanoDrop 2000/2000c where it is recommended that water be used as the blank. • A standard curve can be generated using a minimum of two points which may include two standards or a reference (Protein Pierce reagent/assay buffer only – no protein) and one standard. • The multi-point curve capability allows for multiple replicates for up to seven standards. There is no set order in which standards must be run.
Section 3- Applications -Select the file drop-down option Use current settings as default as a convenient way to limit set-up time for each new workbook. 4. Select Overlay spectra to display multiple spectra at a time. 5. Establish a blank using the appropriate buffer. It is advisable to use the dye reagent and protein buffer without any protein added as both the blank and zero reference sample. -Pedestal Option: Pipette 2 µL of blank solution onto the bottom pedestal, lower the arm and click Blank.
Section 3- Applications Cell Cultures Overview 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.
Section 3- Applications Unique Screen Features The right pane displays features specific to the Cell Cultures application. Task bars in the left pane not described below are described in “Software Overview.” The spectral display shows data for the current sample normalized to a 1 mm path if measured using the pedestal mode or the selected pathlength if using the cuvette option (model 2000c only). The pathlength information is indicated on the Y axis.
Section 3- Applications 4. Establish a blank using the appropriate buffer. The reference or blank solution generally is the buffer that the molecule of interest is suspended or dissolved in. This solution should be the same pH and of a similar ionic strength as the sample solution. - Pedestal Option: Pipette 2 µL of the appropriate blanking solution onto the bottom pedestal and click Blank.
Section 3- Applications Application FAQs Q: Does the NanoDrop 2000/2000c produce results for a continuous spectrum or just selected wavelengths? A: Continuous spectrum from 190-840 nm. The range will vary for specific applications. Q: What are the sample size requirements for the NanoDrop 2000/2000c pedestal? A: As little as 1 µL is usually sufficient for most applications.
Section 3- Applications Q: I am using a colorimetric method (e.g. Bradford, BCA etc.,) to determine the protein concentration. Can I measure the sample using the A280 method on the NanoDrop instrument? A: The Protein A280 application is most applicable to purified proteins. Colorimetric assays such as BCA, Pierce 660 nm, Bradford, and Lowry are generally used for uncharacterized protein solutions and cell lysates.
Section 4- Method Editor 4. Method Editor The Method Editor application and the List of Custom Methods box on the main menu are used to create, edit, save and view custom methods. The Editor Options task bar will be displayed when the Method Editor application is selected and is used to set limits prior to utilizing the Method Editor.
Section 4- Method Editor Left Pane When the Method Editor task bar is selected, the following icons will appear at the top of the left pane: • New - creates a new custom method. • Save - saves a new custom method. This icon will be grayed out until the minimum setting requirements for a new method have been entered. A new custom method may be saved to Groups other than Classic by using the Copy to another group icon located under the method list drop down box.
Section 4- Method Editor • Additional Measurements - adds user-selected or user-defined formulas for additional processing of data. Refer to “Creating New Formulas” below for additional details regarding how to build a custom formula. • Instrument settings - selects the spectral display wavelength range for the method. The auto pathlength option automatically uses the optimal pedestal pathlength for high concentration samples.
Section 4- Method Editor Example: Lysozyme (A(280)* 10 / 26.4) / Path() mg/ml The above example will result in a “Lysozyme” column to be added to the report. The value will be the absorbance at 280 nm multiplied by an adjustment factor of 10 divided by the constant 26.4 divided by the pathlength used in centimeters. Note: The 10 fold factor is used to adjust the E1% extinction coefficient value to a 0.1% value. This adjustment allows the protein concentration to be reported in units of mg/ml.
Section 4- Method Editor Sharing of Custom Methods Custom methods may be shared by simply pasting a copy of the desired method into the appropriate folder. The method folder is located at: XP C:\Documents and Settings\All Users\Shared Documents\Thermo\NanoDrop2000\Custom Methods Vista C:\Users\Public\Documents\Thermo\NanoDrop2000\Custom Methods\Classic Making Custom Method Measurements 1. Select the custom method from the main menu custom method list box.
Section 5-Kinetics 5. Kinetics The NanoDrop 2000c can be used to make time-based kinetic measurements. The Kinetics Editor is used to create, edit, save and view custom kinetic methods. Measurements can also be made without saving a method. The software does not support kinetics measurements using the pedestal mode. Screen Features Left Pane When the Kinetics Editor is selected, the following icons will be displayed at the top of the left pane: • New - creates a new custom method.
Section 5-Kinetics Right Pane When Kinetics Editor is selected, clicking on the icon at the top left will start a new method. A two-step wizard style interface will appear at the bottom of the right pane. This wizard style interface includes Previous, Next, Finish and Cancel buttons on the bottom right of the page. The right pane features include: • Kinetics Tab - displays the current method name, description, result label and time units. The default setting for time units is seconds.
Section 5-Kinetics Data Acquisition Page There are two graphical displays in the right pane of the Kinetics screen: • The Wavelength table to the right of the display shows the absorbance values at each stage of the measurement for all of the wavelengths being monitored. - • The spectrum plot at the bottom of the display shows a trace for each measurement taken in each stage. The data is displayed with absorbance as the vertical axis and wavelength as the horizontal axis.
Section 5-Kinetics Note: Rate vectors are by default locked to the curve and will display the best fit for the two time points selected. Highlight the vector and right-click to deselect the Lock to curve feature to manually move the vector. The data for the rate vector is shown in a table to the right of the Rate display. Fields include: • Vector Name - name of the rate vector. • Time 1 - beginning time of the rate vector. • Time 2 - end time of the rate vector.
Section 5-Kinetics - It may take between 1- 10 minutes for the cuvette holder to reach temperature. - The heater will stop heating when another, workbook, application or method is opened. 4. Establish a blank using the appropriate buffer. Insert a cuvette with the blank solution into the instrument, noting the direction of the light path indicated by the etched arrow and choose Blank. 5. The software will prompt for a workbook name and storage location.
Section 6- Warranty and Maintenance 6. Warranty and Maintenance Warranty All NanoDrop spectrophotometers and accessories manufactured by Thermo Fisher Scientific are under warranty 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. More information about the various plans may be found on our website.
Section 6- Warranty and Maintenance Maintenance of the Sample Retention System Cleaning The primary maintenance requirement for the NanoDrop 2000/2000c is keeping the measurement pedestal surfaces clean. Upon completion of each sample measurement, wipe the sample from the upper and lower pedestals with a clean, lint-free lab wipe to prevent sample carryover and avoid residue buildup.
Section 6- Warranty and Maintenance Pedestal Reconditioning The Bradford reagent as well as other buffers containing surfactants may “un-condition” the measurement pedestal surfaces so that the liquid column does not form well with 1 µL samples. 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.
Section 6- Warranty and Maintenance Solvent Compatibility The NanoDrop 2000/2000c Spectrophotometer pedestals are compatible with most solvents typically used in life science laboratories. These include: methanol, ethanol, n-propanol, isopropanol, butanol, acetone, ether, chloroform, carbon tetrachloride, DMSO, DMF, Acetonitrile, THF, toluene, hexane, benzene, sodium hydroxide, sodium hypochlorite (bleach), dilute HCl, dilute HNO3, dilute acetic acid.
Section 6- Warranty and Maintenance Maintenance FAQs Q. How do I clean the pedestal? A: Add 2-3 µL of water to the bottom pedestal surface and lower the arm to form a liquid column. Raise the arm and wipe both measurement surfaces with a dry, lint-free laboratory wipe. Do not use detergents or isopropanol as cleaning agents as their use may result in the pedestals becoming unconditioned.
Section 7- Diagnostics and Troubleshooting 7. Diagnostics and Troubleshooting The Diagnostics task bar accesses both the Intensity Check and the Calibration Check. The Intensity Check confirms that the internal spectrometer is functioning as expected and the Calibration Check confirms that the instrument is performing within the pedestal pathlength calibration specifications. Intensity Check With the sampling arm down, click Measure to perform an Intensity Check.
Section 7- Diagnostics and Troubleshooting Calibration Check It is recommended that a pedestal calibration check be performed every six months to verify that the instrument is performing within specifications. A vial of Thermo Scientific CF-1 calibration fluid is required to run the calibration check procedure. CF-1 is an aqueous potassium dichromate (K2Cr2O7) solution used to confirm the pathlength accuracy of NanoDrop 2000/2000c spectrophotometers.
Section 7- Diagnostics and Troubleshooting 7. Follow the on-screen prompts in the Customer Guidance text box. Using individual 1µL aliquots of the CF-1 Calibration Fluid, measure 10 replicates. After the 10th measurement, the calibration check results will be displayed on-screen in the Customer Guidance text box. 8. If the instrument does not pass the calibration check using 1 µL samples, immediately repeat the procedure again using 2 µL samples.
Section 7- Diagnostics and Troubleshooting 1. Exit the software. 2. Disconnect the instrument power cord and USB cable. 3. Reconnect the instrument’s power cord, then the USB cable. 4. Restart the software. If the error persists, contact Technical Support. Outside of the US and Canada, please contact your local NanoDrop products distributor. Low Signal Errors Some error messages (e.g.
Section 7- Diagnostics and Troubleshooting If the warning persists and the user visually confirms that the liquid column is forming, perform a calibration check. If the instrument is out of calibration, contact Technical Support. Outside of the US and Canada, please contact your local NanoDrop products distributor.
Section 7- Diagnostics and Troubleshooting Note: Do not use a squirt or spray bottle to apply de-ionized water. • Use a 1.5-2 µL sample volume. Unexpected results can occur when the liquid sample column is not completely formed during a measurement. During the measurement, visually confirm that the liquid column is formed. If necessary, use 1.5 to 2.0 µL sample volume to ensure proper column formation.
Section 7- Diagnostics and Troubleshooting • Wavelength accuracy of the spectrophotometers Although the absorbance of a nucleic acid at 260nm is generally on a plateau, the absorbance curve at 280 nm is quite steeply sloped. A slight shift in wavelength accuracy will have a large effect on 260/280 ratios. For example, a + 1 nm shift in wavelength accuracy will result in a +0.2 change in the 260/280 ratio.
Section 7- Diagnostics and Troubleshooting In addition, application screen captures and Intensity Check .JPGs are of great use in diagnosing problems. Email as attachments to Technical Support. Outside of the US and Canada, please contact your local NanoDrop products distributor. Data Archive Files If you have questions about your data, please send the autosave file containing the data as an email attachment to Technical Support.
Section 7- Diagnostics and Troubleshooting Troubleshooting FAQs Q: Why do I have negative absorbance values? A: A blank measurement was made either using a solution with more absorbance than the sample of interest or on a dirty pedestal. Clean the pedestal and make a new blank measurement with a fresh aliquot of the appropriate buffer.
Section 8-Appendices 8. Appendices Quick Start Guide The Quick Start Guide includes information about a recommended blanking procedure to ensure proper reference measurements. It may be useful to print and post this page near the instrument as a quick lab reference. Software Feature Overview The Software Feature Overview is one page summary of key software features. It may be useful to print and post this page near the instrument as a quick lab reference.
INSTRUCTIONS NanoDrop 2000/2000c Thermo Scientific NanoDrop 2000/2000c Spectrophotometers Quick Start 1. Double click on the desktop NanoDrop™ 2000 software icon and select the application of interest. Follow the prompts for instrument initialization. 2. Ensure Add to report is selected in the left pane to automatically include all measurements in the saved report. 3. Establish a Blank using the appropriate buffer.
INSTRUCTIONS NanoDrop 2000/2000c Software Features The NanoDrop 2000/2000c software interface is divided into a left pane and a right pane. Task bars and Action buttons are located in the left pane while the right pane acquisition pages display the sample spectra. Task Bars Home - displays the main menu with the available selection of applications. Measure (specific application)- active application screen. My Data - manages data archiving and retrieval.
T031‐TECHNICAL BULLETIN N a no D ro p 2000 /2000 c Cleaning and Reconditioning Pedestal Cleaning 1. Apply 3-5 ul of dH20 on to the bottom pedestal. Never use a squirt bottle to apply de-ionized water or any other liquid to the surface of the instrument. 2. Lower the upper pedestal arm to form a liquid column; let it sit for approximately 2-3 minutes. 3. Wipe away the water from both the upper and lower pedestal with a dry, lint-free lab wipe.
