oSpectrometer® nual gEN) manual fluorescence Register your instrument! www.eppendorf.
Copyright © 2014 Eppendorf AG, Hamburg. No part of this publication may be reproduced without the prior permission of the copyright owner. Trademarks Eppendorf® and the Eppendorf logo, Eppendorf BioSpectrometer®, Eppendorf SpectraZoom®, and UVette® are registered trademarks of Eppendorf AG, Hamburg, Germany. Cy® is a registered trademark of GE Healthcare UK Ltd., Buckinghamshire, UK. Hellma® is a registered trademark of Hellma GmbH & Co. KG, Müllheim, Germany.
Table of contents Eppendorf BioSpectrometer® fluorescence English (EN) Table of contents 1 Operating instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Using this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Danger symbols and danger levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of contents Eppendorf BioSpectrometer® fluorescence English (EN) 6 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Selecting a method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Photometry method description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.
Table of contents Eppendorf BioSpectrometer® fluorescence English (EN) 11 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Power supply. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Ambient conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of contents Eppendorf BioSpectrometer® fluorescence English (EN)
Operating instructions Eppendorf BioSpectrometer® fluorescence English (EN) 1 1.1 Operating instructions Using this manual Read this operating manual completely before using the device for the first time. Also observe the instructions for use of the accessories. This operating manual is part of the product. Thus, it must always be easily accessible. Enclose this operating manual when transferring the device to third parties.
Operating instructions Eppendorf BioSpectrometer® fluorescence English (EN) 1.3 Symbols used Depiction Meaning 1. 2. Actions in the specified order Actions without a specified order • List Press this key to perform the described action. or sample Press this softkey to perform the described action.
Operating instructions Eppendorf BioSpectrometer® fluorescence English (EN) 1.
Operating instructions Eppendorf BioSpectrometer® fluorescence English (EN)
Product description Eppendorf BioSpectrometer® fluorescence English (EN) 2 2.1 Product description Main illustration Abb. 2-1: Front and rear view 3 2 3 ce absorban absorban 1 ce height 8.5 m m 1 2 3 abc 4 def 5 gh i jkl 7 pq rs 8 tuv meth od 6 mno 9 wxyz exit func tion 0 del ete µ % ente r standard blan k sample 10 Fig.
Product description Eppendorf BioSpectrometer® fluorescence English (EN) 2.3 Features The BioSpectrometer fluorescence combines two spectroscopic measuring procedures: spectrophotometry and fluorimetry. It is able to carry out both spectrophotometric measurements in the UV/Vis range of 200 nm to 830 nm and fluorimetric measurements at two defined wavelength combinations in the visible range (470 nm excitation/520 nm emission and 470 nm excitation/560 nm emission).
Safety Eppendorf BioSpectrometer® fluorescence English (EN) 3 3.1 Safety Intended use The BioSpectrometer fluorescence is to be used in molecular biology, biochemistry and cell biology research laboratories. The BioSpectrometer fluorescence is exclusively intended for use indoors. All country-specific safety requirements for operating electrical equipment in the laboratory must be observed.
Safety Eppendorf BioSpectrometer® fluorescence English (EN) WARNING! Electric shock due to damage to device or mains cable. Only switch on the device if the device and mains cable are undamaged. Only use devices that have been properly installed or repaired. In case of danger, disconnect the device from the mains supply by pulling the power plug from the device or the mains socket or, by using the isolating device intended for this purpose (e.g., emergency stop switch in the laboratory).
Safety Eppendorf BioSpectrometer® fluorescence English (EN) 3.3.2 Damage to device NOTICE! Damage from the use of aggressive chemicals. Do not use any aggressive chemicals on the device or its accessories, such as strong and weak bases, strong acids, acetone, formaldehyde, halogenated hydrocarbons or phenol. If the device has been contaminated by aggressive chemicals, immediately clean it by means of a mild cleaning agent. NOTICE! Damage to the device from fumigating with aggressive chemicals.
Safety Eppendorf BioSpectrometer® fluorescence English (EN) NOTICE! Damage due to improper cleaning of the cuvette shaft. Only clean the cuvette shaft using a moist cotton swab (see Cleaning on p. 65). Do not allow any liquid to enter the cuvette shaft. Do not reach with your fingers into the cuvette shaft. 3.4 Information on product liability In the following cases, the designated protection of the device may be compromised.
Installation Eppendorf BioSpectrometer® fluorescence English (EN) 4 4.1 Installation Preparing installation Keep the transport carton and the packing material for subsequent safe transport or storage. Check the completeness of the delivery using the information in the delivery package (see Delivery package on p. 11). Check all parts for any transport damage. 4.
Installation Eppendorf BioSpectrometer® fluorescence English (EN) 4.4 Connecting the printer 4.4.1 Thermal printer DPU-S445 Prerequisites Software version 3.4.4.0 or higher is installed on the device. Connect the thermal printer DPU-S445 to the USB port for printers. 1. Connect the printer cable with the USB port for printers 4 (see Main illustration on p. 11). 2. Connect the printer cable with the printer. 3.
Installation Eppendorf BioSpectrometer® fluorescence English (EN) DIP SW-2 Meaning 4 (ON) Zero = Normal 5 (ON) International 6 (ON) Character 7 (ON) Set 8 (OFF) = U.S. DIP SW-3 Meaning 1 (ON) Data Length = 8 bits 2 (ON) Parity Setting = NO 3 (ON) Parity Condition = Odd 4 (OFF) Busy Control = XON/XOFF 5 (OFF) Baud 6 (ON) Rate 7 (ON) Select 8 (ON) = 9600 bps 4.
Installation Eppendorf BioSpectrometer® fluorescence English (EN)
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5 5.1 Operation Overview of operating controls Abb. 5-1: Control panel of the BioSpectrometer fluorescence Fig. 5-1: Key: Control panel of the BioSpectrometer fluorescence Function Keypad: Enter digits and text. Keys 1 to 9 as well as 0: When entering text, next to numbers you also can enter letters and special characters by pressing the key several times. Alternatively, you can switch to a displayed keyboard with the [Keyboard] key.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) Key: Function Move the cursor to the left, right, up, down. • Navigation between input fields. • and keys inside an entry field: Navigate within the character string. • and keys in a result display: Navigate between the sample results of the series of measurement. • and keys within a graph: Navigate on the x-axis of the graph, e.g. for displaying the wavelength-dependent absorbance values in a scan.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5.1.1 Entering text You can enter texts when assigning method names and result units. Restriction: Only digits, letters and the underscore "_" are allowed for method names. Entry via keyboard: Use the and cursor keys to navigate within the entry field and to change single positions in the name. Softkeys: • [Keyboard]: Display keyboard. • [abc]: Change between upper and lower case letters when making entries with the keypad.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5.2 Inserting the cuvette Standard rectangular glass or plastic cuvettes can be inserted in the cuvette shaft: • External dimensions: 12.5 mm × 12.5 mm • Height of light path: 8.5 mm higher than cuvette base • Total height: min. 36 mm The cuvettes must be optically transparent for the respective measuring wavelength.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) The direction of the light path is marked with an arrow on the housing. • Photometry: The direction of the light path from back to front is marked on the housing: "absorbance". • Fluorimetry: The direction of the light path from right to left and back is marked on the cuvette shaft cover: "fluorescence". Abb. 5-2: Marking of light paths fluorescen ce absorban ce height 8.5 m m Fig. 5-2: Marking of light paths 1.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5.3.2 Measuring procedure 5.3.2.1 Selecting a method Use the cursor keys to select the desired method and call up the method with the enter key. For an overview and a detailed description of the methods, refer to the next chapter (see Methods on p. 31). Wizard: The wizard at the top of the display will take you through the method procedure step-by-step.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5.3.2.3 Measuring the blank and standards For evaluations without standards (e.g. DNA measurements), this method step is omitted. 1. Start by measuring a blank (blank key). 2. Then measure all standards one by one (standard key). The display always marks the standard that is to be measured next. Use the [Graph] resp. [Table] softkey to change the result view. Press [Next] to accept the evaluation calculated from the standard results. 5.3.2.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5.3.2.5 Finalizing the method 1. Press [Finish], to complete the measuring series and return to the method selection. 2. After all measurements have been completed, switch off the device and close the cuvette shaft cover to protect the cuvette shaft from contamination. 5.3.2.6 Optional: process results For some methods, you can postprocess the results in the process results method step.
Operation Eppendorf BioSpectrometer® fluorescence English (EN) 5.3.3 Important measurement instructions Check for each measurement: • For plastic cuvettes: How many consecutive measurements can be reliably carried out in the cuvette? • Measure the cuvette blank value before the sample or standard measurements in order to compensate the cuvette blank value in addition to the reagent blank.
Operation Eppendorf BioSpectrometer® fluorescence English (EN)
Methods Eppendorf BioSpectrometer® fluorescence English (EN) 6 6.1 Methods Selecting a method Methods and method templates are delivered preprogrammed. The two Photometry and Fluorimetry main groups are organized in subgroups. Write-protected methods The most important methods in molecular biology. Parameters can be modified, but the modified parameters must be saved under a new method name.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Tab. 6-2: Fluorimetric methods Routine Fluorimetric nucleic acid and protein measurements with reagents supplied by Invitrogen. (Carrying out these procedures may require a license from Molecular Probes, Inc., Eugene, OR, USA or Invitrogen Corporation, Carlsbad, CA, USA.) Basic • Methods for the evaluation of fluorescence measurements with standard or standard curve/line.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) 6.2.2 Routine method group The methods for the Routine group are preprogrammed as fixed methods. Therefore, a new method name is required after the method parameters in the fixed preprogrammed methods have been modified. Nucleic acids • Determination of the concentration of nucleic acids through measurement at 260 nm and evaluation via factor. • Various nucleic acid methods, such as dsDNA or RNA, are preprogrammed.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) • Correction of the influence of the dye spectrum on the accuracy of the biomolecule measurement is possible. • Partial turbidity correction can be performed via the Background parameter. • Additional information on the purity of the measured materials: Ratios A260/A280 and ratios A260/A230 (ratio values only for nucleic acids), absorbance wavelength spectrum.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) 6.3 Fluorimetry method description 6.3.1 Routine method group The methods for the Routine group are preprogrammed as fixed methods. Therefore, a new method name is required after the method parameters in the fixed preprogrammed methods have been modified. The following preprogramed methods are based on Invitrogen's standard operating procedure for the corresponding reagent.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Calibration curve • Measurement of the RFU values and evaluation with a series of 2 to 12 standards • Various evaluation procedures, including linear regression ("Curve fit") and nonlinear regression, can be selected. • Graphical and tabular display of the standard results. • The last saved standard evaluation can be used. • A method for calibration curve evaluation is preprogrammed. 6.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Parameter Entry Explanation Unit Unit for the concentration result. Selection: In the preprogrammed methods of the Routine group, the mg/mL | μg/mL | ng/ selection is restricted to units that are useful for these methods. mL | pg/mL | μg/μL | mg/dL | μmol/mL | nmol/mL | pmol/mL | pmol/μL | U | U/mL | U/L | % | Abs | A/min In addition, further units are freely programmable in the General Method Parameters/Units function. Max. 7 digits.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Parameter Entry Explanation Factor Value input: Factor. Limit: max. of 6 digits including decimal point. Factor for converting absorbance/RFU values into the concentration. Negative factors can also be entered for the following method groups: Dual wavelength, Factor. For the Dye labels method group the factors are not entered into the method procedure individually.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Parameter Entry Explanation Correct A260 1 Selection: on | off Only for the Dye labels method group. Correction of the influence of the dye spectrum on the absorbance with the measuring wavelength of the biomolecule (260 nm or 280 nm). Some of the dye spectra have a low absorbance at 260 and 280 nm. These absorbances distort the calculations for the nucleic acids or the proteins of these methods.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Parameter Entry FOI Selection: Only for the Dye labels method group. none | dye/kb | pmole/ Display of the FOI in addition to the result of the sample μg measurement. The FOI (frequency of incorporation) is a measure for the number of dye molecules per nucleic acid molecule that are integrated into the nucleic acid. Units are "dye/kb" (dye molecules per 1000 bases) or "pmole/μg" (pmol dye per μg nucleic acid). "None": no FOI calculation.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) A method procedure is composed of a maximum of 5 steps. The currently active step is highlighted visually. After the last step, print & export, of a measuring series, the start of a new measuring series is offered as a next step. It once again starts with the sample measurement. Method step Explanation Check parameters Check method parameters. Carry out changes if required.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Saving the method under a new name: You can save the method in the same folder from which you called up the method or in any folder in the Favorites method group. The name (maximum 20 letters) can be entered using a displayed keyboard ([Keyboard] softkey) or directly using the keyboard (see Entering text on p. 23). After saving you will return to the check parameters display. 6.5.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Graphical view of the standard evaluation. With the and cursor keys, you navigate between the standards to view the results. With more than one replicate per standard, you can switch between the replicate results using and . You can also select individual standards from the graphical display and measure or remeasure them. Softkeys • [Table]: Switch to the table display of the standard results.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Additional data • Upper right; first row: Sample number: Counted sequentially and reset to "1" for each new series of measurements. Sample dilution (if provided) • Upper right; second row: Sample identification (ID) (if provided) • Top left: File name with which the data in the print and export method step can be exported as Excel file (see p. 55). Softkeys • [Dilution]: Enter sample dilution.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) 1. Press the [Edit ID] softkey. 2. Enter the sample ID (up to 12 digits). Alternatives for character input: • Keypad: If the key is pressed several times in a row, the possible entries for this key will be shown consecutively. • Display keyboard with softkey [Keyboard]: Select character with the cursor keys and confirm with enter. Softkeys • [Keyboard]: Display keyboard.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) 6.5.4 Measure samples: Results displays This section contains a display of typical results displays for all method groups and an overview of additional results data, which can be accessed using the [Data] softkey.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Method group Photometry Results display Explanation Routine main group Nucleic acids Results display: • Concentration result with absorbance at the measuring wavelength • If activated in the parameters: Ratios A260/A280 • If activated in the parameters: Scan. Navigate between the measuring points on the graph which are used for the result calculation with and . Additional data ([Data] softkey):.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Method group Photometry Results display Explanation Proteins (with reagent) Results display: • Concentration result with absorbance at the measuring wavelength. • For evaluation with standard series: graph of the standard evaluation with plotted sample result. Dye labels Results display: • Concentration results with absorbance at the measuring wavelength of the biomolecule. • If activated in the parameters: Scan.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Method group Photometry Results display Explanation Analog to Protein direct UV (see above) Results display: • Concentration result with absorbance at the measuring wavelength. Basic main group Factor, standard Calibration curve Analog to Proteins (with reagent) (see above) Results display: • Concentration result with absorbance at the measuring wavelength. • Graph of the standard evaluation with plotted sample result.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Method group Fluorimetry Results display Explanation Proteins Analog to Nucleic acids (see above). Results display: • Concentration result with RFU value at the measurement wavelength • Graph of the standard evaluation with plotted sample result. Basic main group Raw fluorescence Results display: • RFU value at the measurement wavelength • Only for dilution: Additional display of the RFU value before conversion.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) 6.5.5 Process results The sample measurement is followed by two optional steps in the method sequence: process results and print & export. In the process results step, you can postprocess the results for some methods. Example: Changing the spectra section of a scan. As for the result display, you can navigate between the sample results of the measurement series with the and cursor keys and select results for postprocessing. Tab.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) After changes have been made, you can exit the current mode using the two softkeys at right: • [Save]: Save changes and return to the process results method step. • [Cancel]: Cancel and return to the process results method step. After the changes have been saved you can apply them to all samples of the measuring series with [Yes]. 6.5.6 Process results: Options Zoom Press the [Zoom] softkey and select one of the following versions.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Variant [spectra-0]: Same as the [spectra] variant, with one exception: The lower limit of the displayed section of the y-axis always equals "0 A". Variant [free]: User-defined values for interval limits can be entered for both axes. Navigation between the entry fields by means of the cursor keys ( , , ). For all 3 versions, the [reset zoom] softkey will bring you back to the original display of the spectrum.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Dye labels method group: Nucleic acid: • After the molar mass has been entered (in base/ base pairs or in kDa): Convert the concentration result to the molar concentration. • After the sample volume has been entered: Calculate the total amount in the sample. Dye: • After entering the volume of the sample: Calculate the total amount in the sample.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) λ grid: 20 nm, min. Δ Abs: 0.200: The peak is not detected because the predetermined value for min. Δ abs is too high. The difference of the absorbance of the peak and the lowest absorbance in the grid is less than 0.2 A. λ grid: 20 nm, min. Δ Abs: 0.050: The peak is detected. 6.5.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Select samples • Press the [Samples] softkey to call up the sample selection. • Use the cursor keys for navigating and confirm with enter. Softkeys • [Select all]: Select all samples • [De-Sel. all]: Cancel selection. Data export The data will be transferred as Excel files (.xls) and can be read with Excel versions Excel 97 and later. For each of the selected data packets, a worksheet is created in Excel.
Methods Eppendorf BioSpectrometer® fluorescence English (EN) Select data packets Results Primary result data; cannot be selected because they are always transferred. Data Additional results data that are displayed during the measurement using the [Data] softkey. Graph Absorbance-wavelength-spectrum. Graph data The basic numeric data for the graph. "export only": Only available for export, i.e., not for printing.
Methods Eppendorf BioSpectrometer® fluorescence English (EN)
Functions Eppendorf BioSpectrometer® fluorescence English (EN) 7 7.1 Functions Functions of the User main group With the function key or the [Function] softkey, you reach a menu containing functions like device settings or calling up saved results. The functions are structured in 3 columns analog to the method selection. The functions in the User main group are accessible to you.
Functions Eppendorf BioSpectrometer® fluorescence English (EN) Subgroup Device calibration Info 7.1.1 Explanation • Option for checking the spectrophotometer; An Eppendorf filter set is required for this. • Option for checking the fluorescence unit. Open Source Licenses and information on registered trademarks. Results memory In the right column, select the method for which you would like to call up saved results. Confirm with enter.
Functions Eppendorf BioSpectrometer® fluorescence English (EN) If you would like to print or export results, select the data packets. The procedure for print and export and the meaning of the function keys corresponds to the print & export method step. 7.1.2 General method parameters In the right column, select the parameter group for which you would like to edit parameters. Confirm with enter.
Functions Eppendorf BioSpectrometer® fluorescence English (EN) To edit a parameter group, use and to select the parameter which you would like to edit. Confirm with enter. Softkeys • [OK]: Save entry and return to the parameter group selection. • [Cancel]: Return to the parameter group selection without making any changes.
Functions Eppendorf BioSpectrometer® fluorescence English (EN) Parameter Explanation • NA name • Factor • Double-stranded Dyes • • • • • • These parameters are loaded into the method parameters when a dye is selected during the programming of a method in the Dye labels group. Dye name Wavelength Ext.coeff. Factor Corr. A260 Corr. A280 Units • Unit The factor is used to calculate the concentration on the basis of the absorbance.
Functions Eppendorf BioSpectrometer® fluorescence English (EN) Softkeys • [Export] and [Print]: Export to a USB stick or print to a PC using a USB cable (see Print & export on p. 55). • [OK]: Return to the function selection. 7.1.4 Device settings The following settings can be modified: • Language: German, English, French, Spanish, Italian. • Date and time. • Information on the light path display. • Frequency of the automatic self test of the device after switching on.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) 8 8.1 Maintenance Cleaning DANGER! Electric shock as a result of penetration of liquid. Switch off the device and disconnect the power plug before starting cleaning or disinfection work. Do not allow any liquids to penetrate the inside of the housing. Do not spray clean/spray disinfect the housing. Only plug the device back in if it is completely dry, both inside and outside.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) 8.1.1 Cleaning the cuvette shaft cover If you would like not only to clean the directly accessible surface of the cuvette shaft cover, you can remove the cover. Do not soak the cuvette shaft cover in cleaning agent. Clean the cuvette shaft cover as described. 1. Lift the cuvette shaft cover with one hand. 2.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) 5. If the cuvette shaft cover needs to be removed for the disinfection, proceed as follows for the diassembly and assembly (see Cleaning the cuvette shaft cover on p. 66). 6. You can use spray disinfection to disinfect the disassembled cuvette shaft cover. 8.3 Checking the device Requirements • Observe the ambient conditions (see Ambient conditions on p. 83). • Carry out an inspection at approx. 20 °C. Avoid temperature variations (e.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) Please connect the Eppendorf thermal printer if you would like to document the values. Abb. 8-1: Inside of the lid of the filter box (sample) Fig.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) 8.3.1.1 Checking photometric accuracy 1. Select the Spectrometer unit function in the Device calibration group and confirm with enter. 2. Select whether you want to check the wavelength systematic error or photometric accuracy, and confirm with enter. Press [Next >] to switch to the measurement. 3. Follow the instructions on the device display and start by measuring the A0 blank filter, and then the first test filter A1.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) 4. Results display after measuring a test filter for testing photometric accuracy. Measure the other two test filters A2 and A3. 5. Results display after measuring all 3 test filters for testing the photometric accuracy. With the and keys, you can view the results for the different test filters again. Press [Finish] to complete the test. 6. Compare the average values and CV values to the supplied table.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) 8.3.2 Checking the fluorescence unit 1. Select the Fluorescence unit function in the Device calibration group. Confirm with enter. 2. Place the F1 filter in the cuvette shaft. Press the Measure softkey. The device measures the test filter 15 times at 2 emission wavelengths.
Maintenance Eppendorf BioSpectrometer® fluorescence English (EN) • Determination of the random error of the excitation light ("noise") • Determination of the signal level and deviation of the emitted light Select the Perform self-test function in the Device calibration group and confirm with enter. At the end of the self-test, the display shows the message PASSED. If the display shows the message FAILED, the self-test has failed. If this error cannot be corrected (see Error messages on p.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) 9 9.1 Troubleshooting General errors Error Measuring results are imprecise. Possible cause Remedy • Reagent is past its shelf life. Ensure that the reagent is still within its shelf life and properly prepared. • Reagent has not been prepared properly. Use clean demineralized water of adequate quality for preparation if required. • Pipetting is not correct.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) Error The measuring results are not correct. Possible cause • The method has not been programmed correctly. Remedy Ensure that the method parameters are entered correctly. • The standard solution has Ensure that the correct standard is used not been prepared and that the measuring solution for the correctly. standard is prepared correctly. • The absorbance of the reagent is drifting.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) 9.2 Error messages You can exit device displays with error messages using the [OK] softkey. System errors require an evaluation by the Technical Service. These errors are shown in English (System error …). Please contact Technical Service in these cases. Other error messages, for which you can carry out troubleshooting measures, are illustrated in the table below. Problem Self test failed. File export failed.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) Problem Cause Solution A method (or folder, dye, protein, nucleic acid, or unit) with this name already exists. • The name under which the method Assign a different name. was saved has already been used for a different method in the same folder. • The message also appears after editing names already given to a folder or to a nucleic acid (dye, protein, concentration unit) (under General Method Parameter).
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) Problem Cause Solution The ID cannot be set. • Error when entering the sample ID. See information in the help box. Different causes are possible. For the precise cause please see the information in the help box. The dilution cannot be set. • Error when entering the dilution. Different causes are possible. For the precise cause please see the information in the help box. Calculation not possible because of division by zero.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) 9.3 Result flags Warnings and error messages for results are displayed in the bottom right of the help box. The header bar of the Help box is highlighted yellow for warnings and red for error messages. Warnings: Decide whether the result is useful for you while taking the displayed warning into consideration. Error messages: No result is displayed; the reason is shown in the error message.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) Problem Cause Solution The coefficient of determination is <0.8. • For methods with evaluation of standard series via the regression procedure: The coefficient of determination for the regression evaluation indicates a significant deviation of the measuring points from the regression line. • Turbidity of the measuring solution. • Measurements at the limits of the photometric measuring range.
Troubleshooting Eppendorf BioSpectrometer® fluorescence English (EN) Problem Cause Solution Measure again considering the possible causes. At least one of the results is negative. • For methods with several results (e.g., Dye labels). • Measuring solution not prepared correctly. • The incorrect factor has been entered (wrong algebraic sign). The result has more than 6 pre-decimal places. Dilute sample and measure again. • Very high sample concentration.
Transport, storage and disposal Eppendorf BioSpectrometer® fluorescence English (EN) 10 10.1 Transport, storage and disposal Transport Use the original packaging for transport.
Transport, storage and disposal Eppendorf BioSpectrometer® fluorescence English (EN)
Technical data Eppendorf BioSpectrometer® fluorescence English (EN) 11 11.1 Technical data Power supply Power supply 100 V to 240 V ±10 %, 50 Hz to 60 Hz Overvoltage category II Degree of pollution 2 Power consumption Maximum power consumption according to name plate: 25 W Approx. 15 W during operation Approx. 5 W with the display dimmed Permitted mains interruption Approx. 10 ms at 90 V Approx. 20 ms at 230 V Protection class I Fuses T 2.5 A/250 V, 5 mm × 20 mm (2 pcs.) 11.
Technical data Eppendorf BioSpectrometer® fluorescence English (EN) 11.
Technical data Eppendorf BioSpectrometer® fluorescence English (EN) 11.6 Further technical parameters Cuvette material For measurements in the UV: Quartz glass or UV transparent plastic (Eppendorf UVette, 220 nm to 1600 nm) For measurements in the visible range: Glass or plastic material Cuvette shaft 12.5 mm × 12.5 mm, untempered Overall cuvette height Min. 36 mm Height of the light beam in the cuvette 8.
Technical data Eppendorf BioSpectrometer® fluorescence English (EN) 11.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) 12 Evaluation procedure This chapter describes the evaluation procedures available in the method programs as well as the calculation of a dilution using the device software. When comparing the measuring results to the results of other photometers/ spectrophotometers, note that the values may be dependent on the bandwidth of the devices.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) 12.1.3 Cuvette correction All absorbance values which are used for result calculation are standardized to the cuvette layer thickness of 10 mm. If a cuvette with a different path length is used, this path length must be defined in the cuvette parameter. In this case, the measured absorbances are corrected to match measuring results with a cuvette layer thickness of 10 mm before converting them to sample results.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) The factor is either entered directly as a parameter during the "Factor" evaluation procedure or calculated during the "Standard" evaluation procedure (evaluation with a standard concentration): F CS AS F = calculated factor CS = concentration of the standard (enter as parameter). AS = measured absorbance of the standard.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) • Use the "linear regression" procedure for calibration lines. • With curvilinear gradients, test which evaluation procedure (quadratic regression, cubic regression, spline interpolation) produces the function that is most suitable to the standard evaluation.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) The application of the evaluation procedure can be activated in the parameters Correct A260 or Correct A280.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) 12.5.3 Conversion to molar concentrations and nucleic acid quantities The conversion only can be applied to nucleic acids and dye methods with nucleic acids as biomolecule components. It is realized in the process results/More calculations method step. 12.5.3.1 Calculation of amount Application: calculating the amount (mass) of nucleic acid in the total sample volume.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) For ssDNA, RNA, Oligo: MM = calculated relative molar mass; unit: kDa bp = entered number of base pairs per molecule b = entered number of bases per molecule • For dsDNA the calculation of the molar concentration is based on the assumption of a double-stranded nucleic acid. For the ssDNA, RNA and Oligo methods, a single-stranded nucleic acid is assumed.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) 12.6 Special evaluation procedures for the dye methods 12.6.1 Calculating the factor for the dye from the absorbance coefficient For the dye methods the concentration of the dye is calculated using a factor from the measured absorbance (see Evaluation with factor or standard on p. 88). The factor is entered for each dye in the General Method Parameter/Dyes function. Alternatively, you can enter the absorbance coefficient.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) 12.6.3 Conversion to amounts of dye The quantity (dimensions) of dye in the entire sample volume is calculated in the process results/More calculations method step. M C u VP ,total M = calculated total amount (mass) of dye in the sample tube. Unit: pmol. C = dye concentration calculated from the measurement. Unit: pmol/μL. VS, total = total volume of the sample in the sample tube; entered by the user under More calculations.
Evaluation procedure Eppendorf BioSpectrometer® fluorescence English (EN) 12.8 Fluorimetry 12.8.1 RFU values Relative Fluorescence Unit: RFU values are a measure of the measured fluorescence. Unlike the absorbance values in photometry, the RFU values are not comparable from device to device, therefore they always need to relate to known concentration or fluorescence standards. 12.8.2 Blank All RFU values always relate to the last measured blank (value).
Ordering information Eppendorf BioSpectrometer® fluorescence English (EN) 13 Ordering information Order no. (International) Order no. (North America) 6135 000.009 – 6135 000.017 6135000017 6137 000.006 – 6137 000.014 6137000014 6137 928.009 6137928009 6135 011.000 6135 010.004 6135 012.007 6135010004 0013 021.566 952010409 0030 106.300 952010051 0030 106.318 952010069 0030 079.345 0030079345 0030 079.353 0030079353 4308 078.
Ordering information Eppendorf BioSpectrometer® fluorescence English (EN)
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