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NanoPhotometer C40/N50/N60/NP80 User Guide

1. 61 Stored Methods erst eire nro irritare 63 Stored 62 Submicroliter Cell 13 23 29 34 41 48 53 55 57 59 61 T Table 24 Technical Gpecfcatons 14 16 ZENNER eege GO Troubleshooting 66 U Biller te dno E 17 Bor EE 68 EL EE 14 Warranty 70 53 Calculations ccccceccececcececcececeececccucececeeeeceees 54 Measurement Protocol a anaanannnnannnnannnnnnnnnnnnnnnns 53 Wavescan een 55 Calculations ccccceccececcececcecececcececcucececaceeceees 56 Measurement 55 EE 14 19
2. dye A320 DOL A320 Edye W o background and with dye correction DOL Fa AC UR AE A280 Cfaye Amax dje Edye W o background and w o dye correction Amax dye DOL A280 47 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 DOL Amax dye A280 A320 Cfaye Degree of labeling dye per protein ratio Absorbance value at the absorbance maximum of the dye 10 mm path Absorbance at 280 nm 10 mm path Absorbance at 320 nm 10 mm path Extinction coefficient of dye in Extinction coefficient protein factor in Dye dependent correction factor at 280 nm 48 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Table 4 Dye Types Absorbance Max Extinction coefficients and dye dependent correction factors NanoPhotometer Dye Type Absorbance Dye dependent Dye dependent Models maximum ext coeff correction of Dye nm M cm factor at 280 nm Cfaye NP80 N60 N50 C40 0 19 NP80 N60 N50 C40 0 70 5 4 0 41 NP80 N60 N50 C40 0 09 NPBO NGO NSO CAO 0 12 NPBO NGO N5O C40 0 08 NP80 N60 N50 C40 0 46 NP80 N60 N50 C40 0 56 4 0 03 NP80 N60 C40 0 05 4 0 08 NP80 N60 N50 C40 0 05 NP80 N60 C40 0 05 4 0 04 NP80 N60 N50 C40 0 15 NP80 N60 N50 C40 0 30 NP80 N60 N50 C40 0 20 N
3. NNN ENN ENNEN NNN ENN ENNEN NNN ENN ENNEN NN 57 METHOD RO uZ uA AM 57 MEASUREMENT PROTOCOL esee DESSEN EDAD E SERERE SEDES ERR 57 EEN 58 MORE APPS CONCENTRATION ENNEN ENNEN EN NN ENN 58 METHOD RO uz ua AE 59 MEASUREMENTS PROTOCOL 4 esee nennen haa an DEDE SERERE SEDES ESSE SEDES ERR 59 CALCULATIONS 60 MORE APPS STANDARD CURVE ENNEN ENN ENNEN NENNEN OR 60 M OVO R E 60 MEASUREMENTS PROTOCOL ana n ERR 61 GAL CULATIONS E 62 CUSTOM APPS S 62 STORED RESULTS ee 62 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 Qu STORED METHODS e 63 5 PREFERENCES E TSNERNNCL UR 65 65 EE 65 EE 65 WIFI ACCESS POINT 65 ILLUMINATION SAMPLE WINDOW ENEE 65 ENABLE NANOVOLUME 65 DYES 65 6 TROUBLESHOOTING 66 SELF CA
4. Add Concentration New Add up to 20 Concentrations and choose Replicates none 2 or 3 51 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Measure a blank and depending on the replicate setting all concentrations Standard 1 Results will be shown in the results area and if replicates are selected a Repl 1 mean value is shown It is possible to exclude single measurements from curve calculation by switching the toggle switch off mean Note The standard curve cannot be altered any longer once the first Ge sample is measured Repl 1 Repl 2 mean 8 Once Standard curve is created or loaded it will be used for concentration calculations in the method It might be necessary to do a blank measurement Note Use for NanoVolume measurements always a minimum of 1 ul for any blank measurement 9 Apply sample and press the sample button to initiate the measurement Note Once the sample measurement is initiated it is not possible to do changes to the standard curve CALCULATIONS Protein concentration is determined using the standard curve by correlating absorbance values of samples with known concentration to calculate the concentration of the unknown sample In order to maintain accuracy and precision please ensure that the R value of the standard curve is 0 95 or greater KINETICS Please contact the Implen support for further details 52 NanoPhotometer NP80 N60 N50 C40 User Guide Versio
5. Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown If it is desired to heat the sample to 37 use the toggle switch to turn on cell holder heater When the cuvette holder has 37 the color changes to green Note Only available for cuvette applications NP80 and C40 oet desired wavelength range Start and End Wavelength to be scanned for the samples Note If samples with a different wavelength range are selected the graphs are shown on the full scan range of 200 900 nm N50 200 650 nm 58 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 5 EE d 9 10 CALCULATIONS Baseline correction is off as the default and can be enabled by selecting the different values for the correction in the drop down menu 377 nm 604 nm 770 nm N A N50 and 823 nm N A N50 Option to smooth the graph with different boxcars off 1 2 boxcar 11 default 2 boxcar 21 and 3 boxcar 61 Option to set calculate a dilution factor for manual diluted samples Apply the blank ddH20 or buffer to the illuminated sample window for the reference measurement and select blank to initiate the reading Note Use always a minimum of 1 ul for any blank measurement Note he illumination of the sample window can be switched off in the pref
6. 12 CONNECTIVITY 22222 20 22252022222202202020202 20 025282022 2 3 2 1 020202 20 12528 02800 220205249420 0 00 905 14 NANOPHOTOMETER SPECIFICATIONS ENNEN ENNEN ENNEN ENNEN ENNEN ENNEN ENN ENNER ENNEN ENK 15 EE 16 SPECTROPHOTOMETER INSTALLATION NENNEN ORDER 16 SOFTWARE INSTALLATION s eren unen rhum uana RR RR 18 NFOS OVERVIEW Ee 18 REQUIREMENTS AND COMPATIBILITY 52 2 22 2 2 9 200 space Toro cs 18 INSTALLING SOFTWARE ON COMPUTER KEEN ENNER ENEE ENEE 18 INSTALLING NANOPHOTOMETER APP ON TABLET OR SMART 19 FIRST STEPS AND CONFIGURATION WIZARD eren naa h nana uaa ESAE SR SE E ARR RENE ENN 19 3 NANOPHOTOMETER 9 20 APPLICATIONS OVERVIEW 20 Tei el ENTE 22 LEE 22 SDE TABBAR rora N A E EEE EEE TE
7. 5 Toggle switch cells ml is default disabled Enable cells ml to get the cells ml calculated Enter the cell specific factor and multiplier e g 1 OD600 5 x 108 cells ml Correction 1 6 Enter the correction factor to compensate for different optical configurations between this and other instruments Option to smooth the graph with different boxcars off 1 boxcar 11 default 2 boxcar 21 and 3 boxcar 61 Manual 8 Option to set calculate a dilution factor for manual diluted samples Dilution Insert cuvette with the reference sample and select blank button to initiate the measurement Note Use for NanoVolume measurements always a minimum of 1 ul for any blank measurement SAMPLE 10 Insert cuvette with the sample and select the sample button to initiate the measurement CALCULATIONS OD600 Agog cf OD600 Optical density at 600 nm Agoo Absorbance at 600 nm 10 mm path D Dilution factor cf Correction factor for spectrophotometer Cells ml Agog cf multiplier Absorbance 600 nm 10 mm D Dilution factor cf Correction factor for spectrophotometer multiplier Multiplier of sample MORE APPS The More Apps icon located on the home screen opens another menu screen with access to icons for additional applications available on the NanoPhotometer The applications featured in this menu include wavelength concentration wavescan absorba
8. Gz NanoPhotometer NP80 N60 N50 C40 User Manual Version 1 1 0 Software Version 1 1 10558 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Declaration of conformity for the NanoPhotometer C40 N50 N60 NP80 This 1 to certify that the Implen NanoPhotometer conforms to the requirements of the following directives 2014 35 EU Low Voltage Equipment Safety Directive 2004 108 EC EMC Directive IEC 60529 Protection class IP20 2002 95 EC Restrictions on the use of certain Hazardous Substances in Electrical and Electronic Equipment ROHS 2012 19 EU EC Directive on Waste Electrical and Electronic Equipment WEEE 2003 108 EC amp 2008 34 EC By ensuring this product is disposed of correctly you will help prevent potential negative consequences for the environment and human health which could otherwise be caused by inappropriate waste handling of this product 47 CFR Part15 815 107 and 515 109 EN 301 489 1 V1 9 2 Radio and ancillary equipment for portable use portable equipment EUT Operating frequency range 2 4 2 4835 GHz EN 301 489 17 V2 2 1 Electromagnetic compatibility and Radio Spectrum Matters ERM IEC 62133 and UN38 3 Battery certification and transport test Standards to which conformity is declared where relent are as follows IEC EN 61010 1 2012 Safety requirements for electrical equipment for measurement control and laboratory use General requirements EN61326 1 2013 Electroma
9. 10 mm path A320 Absorbance at 320 nm 10 mm path Extinction coefficient protein factor in g cm l D Dilution factor Table 3 Protein extinction coefficients Type g Ext Coeff Mol Ext Coeff M cm BSA SA Mouse SA Human IgG Mouse pe Humar Lysozyme N A N A OD1 1 00 Dye labeled Protein UV280 Concentration MW g mol 66 400 66 000 69 365 160 000 150 000 190 000 3 984 N A For dye labeled proteins the concentration of the protein is calculated using a modified form of the Beer Lambert equation For these calculations the instrument considers the absorption maximum of the dye and a certain dye specific correction factor at 280 nm see 44 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Table 4 The dye concentration is calculated with or without background dye correction for are as follows With background and with dye correction A280 A320 Cfaye Amaxaye A320 With background and without dye correction A280 A320 Without background and with dye correction C A280 Chaye T Amax dye Eprot E 45 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Without background and without dye correction H Concentration in mg ml A280 Absorbance at 280 nm 10 mm path A320 Absorbance at 320 nm 10 mm path Amax dye Ab
10. 80 7 NANO 27 31 18 Nucleic ele TEE 34 36 Measurements Drotocol 34 OCOD GOO mE 50 Uer RUE de LEE 52 Measurements Drotocol 51 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 P Parameter 24 EMEN t 17 Power Adapter 4 12 65 KE 65 E 65 Protein Assay E 49 Measurements 47 Protein ASSAYS 46 Prolein 40 Calculations 1 reete 42 Measurements 41 Rear Panel E 11 Replacement Parts 69 M n 69 eme 69 69 67 Results 24 5 Safety Information 16 Eau EEN 25 Software 67 68 Solvent 32 Standard Ref 60 62 Measurements
11. Encrypted USB flash drives are not compatible Note Cordless Bluetooth mice are not supported Use only corded mice USBB There is a USB B port located on the rear panel of the instrument which is compatible with the USB cable provided to connect the NanoPhotometer to a computer This USB connection can be used to control the NanoPhotometer via a computer LAN There is an Ethernet LAN connection port on the rear panel of the instrument which enables the NanoPhotometer to connect with computers within close proximity This Ethernet connection can be used to transfer data from the NanoPhotometer to a computer To access the directory of the NanoPhotometer via LAN enter the serial number or IP Wserial number MP of the NanoPhotometer in the Windows Explorer Mac OS X Finder and confirm with the enter key Note Plug in the LAN cable before starting the NanoPhotometer Note The maximal LAN cable length is 10 meters Bit rate is 1 Gbit s HDMI There is an HDMI port located on the rear panel of the NanoPhotometer which is compatible with HDMI 1 4 or better cables to connect the NanoPhotometer to HDMI compatible monitors 14 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 NANOPHOTOMETER SPECIFICATIONS NanoVolume Performance Detection Range dsDNA Detection Range BSA Minimum Sample Size Photometric Range t0 mm equivalent Path length dilution factor Vortexer Cuvette Performance Detection Range dsDN
12. When the cuvette holder has 37 C the color changes to green Note Only available for cuvette applications NP80 and C40 The baseline correction is disabled as the default It is possible to choose between different wavelengths for the baseline correction Options are 377 nm 604 nm 770 nm N A N50 and 823 nm N A N50 Curve Fit Select the curve fit type Options are linear regression and zero regression Linear Regression v forces the straight line through the origin Standards gt Standard new no selection possible SECUS New Add Concentration New Add up to 20 Concentrations and choose Replicates none 2 or 3 64 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Measure a blank and depending on the replicate setting all concentrations Standard 1 Absorbances will be shown in the results area and if replicates are selected Repl 1 a mean value is shown It is possible to exclude single measurements from E curve calculation by switching the toggle switch off mean Note The standard curve cannot be altered any longer once the first poroi sample is measured Repl 1 BLANK 7 Once Standard curve is created or loaded it will be used for concentration calculations in the method It might be necessary to do a blank measurement Note Use for NanoVolume measurements always a minimum of 1 ul for any blank measurement 8 Apply sample and press the sample button to initiate
13. 0 QD Table 2 The dye concentration is calculated with or without background correction as follows With background correction _ m D paa ane F Edye 10 Without background correction E Amax dye di Egye 10 Concentration in ng ul Amax dye Absorbance value at the absorbance maximum of the dye 10 mm path A320 Absorbance at 320 nm 10 mm path Save Extinction coefficient of dye in D Manual dilution factor Frequency of Incorporation FOI FOI is the degree of labeling based on dye incorporation in a labeled nucleic acid sample It is generally expressed as the number of dye molecules incorporated per 1000 nucleotides FOI can be calculated with or without background dye correction as follows With background correction with dye correction 324 5 ax de A390 _ dye 10 20 With background correction without dye correction 324 5 nax get A390 FOI 6 x 10 A560 n A320 Bous Without background correction and with dye correction 324 5 Amax dye NEIN 10 A560 Cfaye Amax dye Without background correction and without dye correction 324 5 Amax dye 10 A260 Enuc 38 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 FOI A260 A320 Amax dye Enuc Frequency of Incorporation dye per 1 000 bases Absorbance at 26
14. 1 log logT T 10 Note Corresponding absorbance value e g Absorbance value A 230 etc normalized to 10 mm path length Transmittance Calculations cuvette mode only In the wavelength application it is possible to measure the absorbance A and transmittance on a sample with respect to a reference at a specific wavelength Transmittance is the ratio of light intensity remaining after it has passed through the sample to the initial incident light intensity T lo 57 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 1 x 100 Io MORE APPS WAVESCAN M METHOD OVERVIEW Using the wavescan application it is possible to obtain the complete spectral scan for a defined wavelength range between 200 900 nm NP80 N60 C40 or from 200 650 N50 MEASUREMENT PROTOCOL 1 140 0 07 mm Wavelength Wavelength Select the More Apps icon from the Home screen and then the Wavescan icon from the More Apps screen For NanoVolume application Select the dilution depending on the sample concentration Note There is no automatic path length setting in this method Select either a virtual dilution of 15 path length 0 67 mm or of 140 path length 0 07 mm For cuvette application Select the path length depending on the used cuvette Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume application with submicroliter cell C40 only
15. 47 Bradford Aesay nnns 47 d ERROR 22 C ET 70 CONMD QUID UE 18 Gohvent meme nemen nenne erras 32 Concentration eene eene nnne nennen 58 ONG 60 Measurements Drotocol 59 Connecting CGable 12 CGonnechvity 14 EEN 68 ADDS EE 62 BUTTER 28 31 D 72121120872 mE RTI 26 Didymium Glass Elter 12 EEN 13 E 26 zd RP PIPER 13 G CIE PRI EET TL 24 Graph Overlay EEEE 24 el 8 NEE 14 eet LEE 22 EE 26 Installation EEN 18 Spectrophotometer 16 L I E E E EEA 69 E E 14 Lowry 47 69 Cuvette REDE 28 31 Nano 27 31 More e 52 Absorbance Ratio een 57 Concentration eee nennen nnne 58 Standard Curve ccccccccsceccececcecececcececcecuccececeeceees 60 Wavelength eese 53 55 NanoPhotometer 40 10 NanoPhotometer 50 9 NanoPhotometer 60 8 NanoPhotometer
16. Apply sample to the sample window and press the sample button to initiate the measurement 35 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD CALCULATIONS Nucleic Acid Concentration To determine the concentration of nucleic acids in solution the absorbance as measured at the wavelength of 260 nm is used The function describing the relationship between concentration and absorbance is a modification of the Beer Lambert law equation The concentration of the nucleic acid samples can be calculated with or without background correction depending on if the background correction is turned on for the measurements Withou C Aog t background correction 0 Enuc D With background correction A260 A320 D Table 1 Type dsDNA SSDNA miRNA Oligo miRNA A320 Concentration in ng ul Absorbance at 260 nm 10 mm path Absorbance at 320 nm 10 mm path Manual dilution factor Extinction coefficient nucleic acid factor in ng cm ul Nucleic acids extinction coefficients c 50 ng cm ul 37 ng cm ul 40 ng cm ul 33 ng cm ul 33 ng cm ul Seq calculated via extinction coefficient of constituent nucleotides entered Oligo Seq calculated via extinction coefficient of constituent nucleotides entered Custom Option to enter any factor between 15 and 150 ng cm ul For dye Dye labeled Nucleic Acid Concentration labeled nucleic
17. Ext Coefficient enter Ext Coefficient default 0 667 l g cm SA Human IgG Mouse IgG Human IgE Human 001 Mol Ext Coeff Ext Coeff 3 For NanoVolume application Select the volume of sample to be applied Note 1 2 ul default automatic path length change 0 3 ul measures only the 0 07 mm path length possible for samples with concentrations e g BSA gt 12 6 mg ml For cuvette application Select the path length depending on the used cuvette Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter 42 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown 4 The background correction is enabled as the default It is possible to disable the background correction with the toggle switch 5 If itis desired to heat the sample to 37 C use the toggle switch to turn cell holder heater When the cuvette holder has 37 C the color changes to green Note Only available for cuvette applications NP80 and C40 6 If a dye labeled sample is used please add a dye by tapping on the Add Dye Label option and select a dye from the drop down list There is also an S option for dye correction which can be en disabled with a tog
18. GB Onboard with built in touchscreen computer smart phone and tablet Windows 7 32 and 64 bit Windows 8 32 and 64 bit OS X iOS and Android 4 inch screen Apple iPad 2 iPhone5 and iOS 6 Android version 4 4 Quadcore 1 2 GHz with 1 GB RAM 15 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD General Specifications Size 200 mm x 200 mm x 140 mm Weight 3 8 5 2 kg depending on configuration Operating Voltage 90 250 V 50 60 Hz 60 W 90 W with battery pack 18 19 VDC Display 1024 x 600 pixels touchscreen glove compatible Certifications IEC EN 61010 1 2012 and EN 61326 1 2013 Battery Pack optional rechargeable lithium ion battery 95Wh 6 6Ah Operation time up to 8 h min charging cycles 800 Battery Certification IEC 62133 and UN38 3 transport test In and Output Ports 2 USB A USB B HDMI Ethernet WLAN Additional Data Input Mouse and keyboard options security Slot for Kensington lock Features and specifications are subject to change without notice US Patents 20080204755 and 20080106742 Windows is a trademark of Microsoft Mac OS amp iOS are trademarks of Apple Android OS is a trademark of Google Linux is a trademark of Linus Torvalds 2 GETTING STARTED SPECTROPHOTOMETER INSTALLATION Safety Information Before commencing installation please take time to familiarize yourself with warning labels and symbols on your instrument and their meaning These are to inform you where p
19. Of v 7 Option to smooth the graph with different boxcars off 1 boxcar 11 62 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD default 2 boxcar 21 and 3 boxcar 61 8 Option to set calculate a dilution factor for manual diluted samples 9 Apply the blank ddH20 or buffer to the illuminated sample window for the reference measurement and select blank to initiate the reading Note Use always a minimum of 1 ul for any blank measurement Note The illumination of the sample window can be switched off in the preferences 10 Use a lint free laboratory wipe to clean both the sample window and mirror prior to applying the next sample Note It could be helpful to apply the blank a second time and read it as a sample to ensure a proper blank 11 Apply sample to the sample window and press the sample button to initiate the measurement CALCULATIONS In this method the concentration of the sample is calculated based on the Beer Lambert law given the user specified wavelength of interest and user defined extinction coefficient The equations for calculating concentration with and without background correction are as follows Without background correction C E D C Concentration ng ul An Absorbance at user specified path length n 10 mm path D Dilution factor extinction coefficient factor MORE APPS STANDARD CURVE METHOD OVERVIEW The construction of a calibration curve from multiple
20. application For best results use the cuvette mode NP80 and C40 models only for OD600 measurements 1 Select the OD600 icon on the home screen 2 For Cuvette Application Select the path length depending on the used cuvette I Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume Application Select the dilution depending on the sample concentration Note There is no automatic path length setting in this method Select either a virtual dilution of 15 path length 0 67 mm or of 140 path length 0 07 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown 3 If itis desired to heat the cuvette compartment to 37 turn the toggle switch on for heated cuvette Once the cuvette reaches the desired temperature the switch will turn from orange to green Wavelength 4 Default wavelength is 600 nm but can be changed depending on the application 54 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD
21. area Options are BCA Assay Bradford Assay Lowry Assay Biuret Assay Biuret Bradford Lowry For Cuvette Application Select the path length depending on the used cuvette Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume Application Select the dilution depending on the sample concentration Note There is no automatic path length setting available in this method Select either a virtual dilution of 15 path length 0 67 mm or of 140 path length 0 07 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown 4 If itis desired to heat the sample to 37 C use the toggle switch to turn on cell holder heater When the cuvette holder has 37 C the color changes to green Note Only available for cuvette applications NP80 and C40 The baseline correction is disabled as the default It is possible to choose between different wavelengths for the baseline correction Options are 377 nm 604 nm 770 nm not N50 and 823 nm not N50 Curve Fit Select the curve fit type Options are linear regression and zero regression Linear Regression v forces the straight line through the origin Standards otandard new no selection possible New mm n SE
22. at this time the user needs to define if the samples should be saved or deleted Change o Cuvetia The Change to Cuvette button NP80 is located at the button of the parameter window The change to cuvette nanovolume button toggles between nanovolume and cuvette applications Selecting the Change to Cuvette button activates the cuvette compartment opens the sliding cover and switches the red arrow LED on Only cuvette measurements are possible when this mode is selected Selecting the Change to Cuvette button while it is activated will deactivates the cuvette compartment closes the sliding cover and switches the LED off and only nanovolume measurements are possible Note Please make sure that the nanovolume arm is closed during cuvette measurements The Change to NanoVolume button C40 is only shown when submicroliter cell applications are enabled The Change to NanoVolume Cuvette button toggles between cuvette and submicroliter cell applications With the NanoVolume submicroliter application the parameter of each method show a dropdown for lid selection Lid 5 10 50 100 and 250 Choose the correct lid and the results are automatically calculated considering the virtual dilution SIDE TAB BAR On the left side of the measurement screen there is a vertical tab bar that contains four tabs including parameters data graph and table The different tabs allow organizing the measurement screen It is possible to show or hide the differe
23. concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown If it is desired to heat the sample to 37 use the toggle switch to turn on cell holder heater When the cuvette holder has 37 C the color changes to green Note Only available for cuvette applications NP80 and C40 The background correction is enabled as the default It is possible to disable the background correction with the toggle switch If a dye labeled sample is used please add a dye by tapping on the Add Dye Label option and select a dye from the drop down list There is also an option for dye correction which can be en disabled with a toggle switch Added Dyes be deleted by tapping on the delete icon 99 Note If the used dye is not available in the drop down list please go to preferences and add a custom dye to the dye list Option to set calculate a dilution factor for manual diluted samples Apply the blank ddH20 or buffer to the illuminated sample window for the reference measurement and select blank to initiate the reading Note Use always a minimum of 1 ul for any blank measurement Note The illumination of the sample window can be switched off in the preferences Use a lint free laboratory wipe to clean both the sample window and mirror prior to applying the next sample Note It could be helpful to apply the blank a second time and read it as a sample to ensure a proper blank
24. of 2 0 ul Standard Cuvette applications can be performed with 10 mm 5 2 mm and 1 mm path length quartz glass or plastic cuvettes with a center height of 8 5 cm Note NanoVolume application in the NanoPhotometer C40 is only available with the optional accessory submicroliter cell from Implen NANOVOLUME MEASUREMENT Basics N50 N60 NP80 1 Select a method depending on your sample and set the parameter for the measurement 2 Ensure that the sample window and the mirror in the sample arm are clean Use the integrated vortexer N60 and NP80 only to mix your sample to achieve an homogenous sample 4 Raise the sample arm and pipette the appropriate amount of sample onto the illuminated sample window The illumination turns automatically off lowering the arm d aeo Note Do not overfill the well Note Save low energy red light LED 5 Lower the sample arm and initiate a blank measurement with the blank button 27 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 6 Clean the measurement window and mirror on the sample arm with a slightly wet lint free tissue Use water 70 ethanol or isopropanol Note Make sure that the metal contact face around the measurement window and the mirror is clean Note Do not use aggressive solvents such as strong acids or bases or organic solvents at any time If unsure please contact support implen de for detailed information about your specific rea
25. programed methods it is also possible to manually enter the specific for the protein of interest using the Mol Ext Coefficient or Ext Coefficient protein option For correct calculation it is necessary to supply either the mass extinction coefficient in l g cm or b or the molar extinction coefficient in M and the molecular weight of the protein expressed in molar mass units g mol To determine the degree of dye labelling of a protein the absorbance measured at the wavelength corresponding to the absorbance maximum of the fluorescence dye is used see Table 3 The corresponding extinction coefficient of the dye is used along with the in the Beer Lambert Law to determine the dye concentration Note It is important to ensure the extinction coefficient and units entered are correct in order to ensure that calculations are performed properly for accurate concentration values MEASUREMENTS PROTOCOL 1 Select the Protein UV icon on the home screen To change between NanoVolume and cuvette application NP80 C40 only use the Change to Cuvette NanoVolume button below the parameter area 2 Select the protein type in the drop down of the parameter area Options are BSA SA Mouse SA Human IgG Mouse IgG Human IgE SR AER Human Lysozyme OD1 Mol Ext Coefficient Ext Coefficient e For Mol Ext Coefficient enter Molecular Weight default 66 400 g mol and Mol Ext Coefficient default 44 289 M For
26. slit monochromator optics slit geometry etc different spectrophotometer types will tend to give different OD 600 readings for the same turbid sample Therefore if results from different spectrophotometers are to be compared they must be normalized first using appropriate calibration curves A calibration curve can be constructed by comparing measured OD 600 to expected OD 600 Expected OD 600 is determined by counting cell number using an alternative technique for example microscope slide method and converting to OD 600 using the rule of thumb that 1 OD 600 5 x 10 cells ml for E coli The NanoPhotometer comes with a correction factor of 1 as default To compare OD 600 values between different spectrophotometers it is necessary to determine the constant deviation or ratio between the absorbance values for the same sample from each instrument and use this factor within the setting correction factor of your NanoPhotometer Software Note The use of 10 mm path length disposable cells is recommended for optical density measurements of cell culture solutions Also to prevent the suspension settling too quickly and giving an OD reading that changes with time glycerol should be added to the sample 53 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Stationary Decline Log Phase Figure 1 Bacterial growth curve MEASUREMENTS PROTOCOL Note It is not recommended to use the NanoVolume application for the OD600
27. the cuvette and ensure that the filling volume is sufficient to allow light path to pass through the solution 4 Insert cuvette into the cell holder 5 Initiate a blank measurement with the blank button Upon completion of measurement remove the cuvette 6 Add sample to cuvette and ensure that the sample volume is sufficient to allow light to pass through the sample 7 Initiate a sample measurement with the sample button Upon completion of measurement remove the cuvette 8 Apply further samples SUBMICROLITER CELL MEASUREMENTS Basics C40 The Implen submicroliter cell can be used with the NanoPhotometer C40 Insert the submicroliter cell to the cell holder with the cell windows facing the light beam We recommend facing the Implen logo to the right side The light beam is directed from BACK to FRONT as indicated with the white arrows Note For best performance insert the submicroliter cell always in the same direction 1 Select a method depending on your sample 2 Enable the NanoVolume application with the Change to NanoVolume button below the parameter area Note If there is no Change to NanoVolume button available in the method go to preferences general and enable the NanoVolume option We guarantee our submicroliter 29 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD cell specifications only for Implen submicroliter cells which are aligned to the NanoPhotometer 40 by Implen 3 Set the p
28. the data will be transferred to the device that is currently connected with the NanoPhotometer Delete Selecting the delete icon opens a full screen overlay dialog window with delete options Concentrat A260 260 280 260 230 Select files EE Toggle switch delete selected Delete Toggle switch delete all Delete button Delete selected This option deletes the current selection of data in the table If the delete selected toggle switch is green the delete all toggle switch will automatically be deactivated Delete all The delete all button allows the user to delete all of the data from the current method If the save all toggle switch is green the save selected toggle switch will automatically be deactivated 26 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Once the delete button is selected a message will appear asking Do you want to delete all selected files selecting cancel will return to the delete menu screen and selecting delete will delete the selected data Note The delete function is not available for the software version designed for smart phones BASIC OPERATION The NanoPhotometer product line offers a complete solution for nanovolume N50 N60 NP80 C40 and standard volume C40 NP80 applications With the NanoPhotometer nanovolume applications the sample volume ranges from 0 3 ul to maximum sample volume
29. your supplier 72 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD CLEANING AND GENERAL CARE Switch off the NanoPhotometer and disconnect the power cord prior to external cleaning Use a soft wet cloth or dry microfiber cloth to clean all external surfaces A mild liquid detergent may be used to remove stubborn marks Approved disinfectant solutions include Apesin disinfection spray Tana Chemi GmbH Incidin Liquid amp Inciddin Foam Ecolab and Lysoformin Spezial Lysoform Dr Hans Roseman GmbH Note Observe all necessary precautions if dealing with hazardous samples or solvents 9 WARRANTY Implen guarantees that the product supplied has been thoroughly tested to ensure that it meets its published specification The warranty included in the conditions of supply is valid for 12 months and is valid only if the product has been used according to the instructions supplied Implen or your supplier can accept no liability of loss or damage arising from the faulty or incorrect use of this product 73 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 10 ALPHABETICAL APPENDIX A Absorbance Hato ener 57 eU zie a cO 58 Measurement Protocol esse 57 ACceseorles 11 13 LE Te EEN 66 WAINTONMANICE 67 Report Problem 67 S133216 EE 67 17 69 BCA ASSAY 47 Biuret Assay
30. 0 60 Hz power supply system For Mobile models please load the battery pack at least for 3 hours prior to the first use Turn the instrument on using the power button on the rear panel after it has been plugged in The instrument will perform a series of self diagnostic checks Please read the complete user manual prior to use Please contact original supplier immediately if technical or sample handling difficulties are experienced Note If this equipment is used in a manner not specified or in environmental condition not suitable for safe operation the protection provided by the equipment may be impaired and the instrument warranty voided 17 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD SOFTWARE INSTALLATION NPOS OVERVIEW NPOS is a Linux based operating system designed for the NanoPhotometer NPOS can store data either to a common directory or be configured to save to independent directories according to file format and or instrument NPOS can save data in an Implen IDS format or as an Excel format file Note Please do not connect the instrument to a computer until the NanoPhotometer NPOS software is installed on the computer REQUIREMENTS AND COMPATIBILITY The NPOS user interface is designed that all features can be operated by using a touchscreen If the software is installed on a computer without touchscreen the user interface can be operated by using a keyboard and a mouse Before starting the ins
31. 0 nm 10 mm path Absorbance at 320 nm 10 mm path Absorbance value at the absorbance maximum of the dye 10 mm path Extinction coefficient of dye in Extinction coefficient nucleic acid factor in ng cm ul Dye dependent correction factor at 260 nm 39 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Table 2 Dye Types Absorbance Max Extinction coefficient and dye dependent correction factors NanoPhotometer Dye Type Absorbance Dye dependent Dye dependent Model maximum of ext coeff correction factor Dye nm in at 260 Cfaye NP80 N60 N50 C40 345 0 25 NP80 N60 N50 C40 492 0 30 NP80 N60 N50 C40 525 0 24 NP80 N60 N50 C40 555 0 21 NP80 N60 N50 C40 555 0 04 NP80 N60 N50 C40 576 0 45 NP80 N60 N50 C40 588 0 43 NP80 N60 C40 650 0 00 NP80 N60 C40 680 0 00 NP80 N60 N50 C40 550 0 08 NP80 N60 N50 C40 581 0 08 NP80 N60 C40 Cy5 649 0 05 NP80 N60 C40 675 0 05 NP80 N60 N50 C40 503 0 29 NP80 N60 N50 C40 553 0 05 NP80 N60 N50 C40 560 0 03 NP80 N60 C40 649 0 05 NP80 N60 C40 653 0 04 NP80 N60 N50 C40 Texas Red 9 85 000 0 23 Ratios utilizing nucleic acids often require minimum purity standards Common contaminants of nucleic acid samples include proteins organic compounds and other Based on the common contaminants of nucleic acid samples the 260 280 and 260 230 ratios are calculated for nucleic acids to give an indication of the purity
32. A Detection Range BSA Photometric Range Center Height Z Height Cell Types Heating Optical Specifications Wavelength Scan Range Time for Full Scan Range Wavelength Reproducibility Wavelength Accuracy Bandwidth Stray Light Absorbance Reproducibility Absorbance Accuracy Zero Stability Noise Optical Arrangement Lamp Lifetime 1 ng ul to 16 500 ng ul N50 5 ng ul 7 500 ng ul 0 03 mg ml to 478 mg ml N50 0 15 mg ml to 217 mg ml 0 3 ul 0 02 330 A N50 0 1 150A 0 67 and 0 07 mm 15 and 140 2 800 rpm tube size up to 2 0 ml 0 1 ng ul to 130 ng ul 0 003 mg ml to 3 7 mg ml 0 2 6A 8 5 mm outside dimension 12 5 x 12 5 mm 37 C 0 5 200 900 N50 200 650 nm 3 5 6 0 seconds 0 2 nm N50 1nm 0 75 nm N50 1 5 nm 1 8 nm N50 5 nm 0 596 at 240 nm using Nal N50 296 and 196 at 280 nm using Acetone N50 2 0 002 A 0 67 mm path 280 nm N50 0 004 A 0 67 mm path 280 nm 1 75 96 9 0 7 A 0 67 mm path 280 nm of the reading 0 003 A hour after 20 min warm up 280 nm 0 002 A rms at 0 A 280 nm 0 002 A pk to pk at 0 A 280 nm 1 x 3648 CCD Array N50 1 x 1024 CCD Array Xenon flash lamp 10 flashes up to 10 years Processing Power and Compatibility Operating System Onboard Processor Internal Storage Control Options ooftware Compatibility Min Requirement Smart phone Tablet Linux based OS Quad Core 1 GHz 8
33. A 40 MEASUREMENTS PROTOCOL eege NENNEN NENNEN NENNEN NENNEN ENNEN 41 e gel 42 NNEN 46 ageleet 46 MEASUREMENTS 47 EEN 49 KINETICS 49 00600 50 METHOD e TEEN 50 MEASUREMENTS PROTOCOL aia wr er Nba i xr VR R8 51 UL Pelle I MRNCNONEL E W w B t 52 MORE AFPS 52 MORE APPS WAVELENGTH ENN ENNEN ENNEN NENNEN ENNEN NNN ENN ENNEN NNN ENN ENNEN NNN ENN ENNEN NN 53 METOD OVERVIEW ee 53 MEASUREMENT PROTOCOL nnana 53 CALCULATIONS 54 MORE APPS WAVESCAN rss 55 e 55 MEASUREMENT PROTOCOL 55 EEN 56 MORE APPS ABSORBANCE RATIO
34. EA EAA E EA A E AEAEE 23 DATA PROCESSING DIALOGS SE AERA 25 BASIC nn 27 NANOVOLUME MEASUREMENT BASICS 50 60 27 CUVETTE MEASUREMENT BASICS C40 NP80O 2 eese eere rre een nennt nnn 28 SUBMICROLITER CELL MEASUREMENTS BASICS C40Q 2 2 elec cler iren ee sana sauna aa nhan RE nha 29 SAMPLE HANDLING ENKEN ENEE ERES ENEE ENNEN ENKEN ENEE 31 BATTERY OPERATION ee 33 4 NANOPHOTOMETER APPLICATIONS Ask NN RN NENNEN NENNEN KEN N NNN ENNEN NENNEN NENNEN NENNEN NN ENN AN 34 NUCLEIC ACDS E 34 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD METOD OVERVIEW E O 34 MEASUREMENTS EE 34 e Fe kel 36 PROTEIN OV A ec 40 mm
35. IQ OQ documentation is also available Please contact your local Implen office or an authorized Implen partner for further information Support agreements that help to fulfill the demands of regulatory guidelines concerning GLP GMP include calibration certification using filters traceable to international standards during production and quality control certified engineers and calibrated test equipment approved to ISO 9001 standard automatic self diagnostic calibration test during start of the NanoPhotometer result is documented in each data output file and possibility to save a Implen Document Source IDS file no data manipulation possible REPLACEMENT PARTS Lamp Replacement The NanoPhotometer is equipped with a xenon flash lamp and should not need replacement until several years of use In the unlikely event the lamp does need to be replaced this should be done by the manufacturer or a certified service engineer from your supplier Battery Replacement The optional battery pack can only be assembled or replaced by the manufacturer or a certified service engineer from your supplier Note Danger of explosion if battery is incorrectly replaced Replacement only with the same or equivalent type recommended by the manufacturer Replacement needs to be done by a certified service engineer Touchscreen Replacement The optional touchscreen can only be assembled or replaced by the manufacturer or a certified service engineer from
36. LIBRATION TEST EN NENNEN NENNEN ENNEN ENN ENNEN ENN ENNEN ENNEN NENNEN ENNEN ENN 66 7 EE 66 67 REPORT PROBLEM eet 67 SOFTWARE MAINTENANCE EE 67 RES EE 67 SOFTWARE UPDATE EE 68 CONTACT 68 8 MAINTENANCE m 69 MAINTENANCE FREE ENNEN ENNEN ENN ENNEN ENNEN ENN ENN ENN ENNEN RR RR 69 REPLACEMENT IP aa EE 69 CLEANING AND GENERAL 70 gt o sub 70 TU ALPHABETICAL APPEND T1 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 IMPLEN 1 NANOPHOTOMETER AT A GLANCE NANOPHOTOMETER OVERVIEW The Implen NanoPhotometer spectrophotometer is a mobile simple to use UV Visible instrument with a CCD array detector with options ranging from nanovolum
37. PBO NGO NSO CAO 0 60 NPBO NGO NSO C40 0 20 NPBO N6OIN5O C40 0 12 NPBO NGO NSO CAO 0 18 NP80 N60 N50 C40 Texas Red 595 80 000 0 18 PROTEIN ASSAYS METHOD OVERVIEW Protein concentration may be measured using colorimetric assays in which certain reagents are added to the protein solution to generate a colored product either a protein cupric ion chelate as in the Biuret Lowry BCA assays or a protein dye complex as in the Bradford assay In these colorimetric assays the absorbance is measured in the visible range at the appropriate wavelength for each assay and compared against a standard curve prepared by serial dilution of a protein standard of known concentration A linear regression analysis of the calibration 49 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD standard data points is calculated by the NanoPhotometer A correlation coefficient in the range of 0 95 to 1 00 indicates a good fit to a straight line Bradford Assay Method depends on quantifying the binding of a dye Coomassie Brilliant Blue to an unknown protein and comparing this binding to that of a standard curve prepared from a set of known protein of known concentrations at 595 nm This standard is usually BSA bovine serum albumin Biuret Assay Method depends on a reaction between cupric ions and peptide bonds in an alkali solution resulting in the formation of a complex absorbing at 546 nm BCA Assay Method depends on
38. a reaction between cupric ions and peptide bonds coupled with the detection of cuprous ions using bicinchoninic acid BCA giving an absorbance maximum at 562 nm The BCA process is less sensitive to the presence of detergents used to solubilize membranes Lowry Assay Method is based on the Biuret reaction Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion Monovalent copper ion and the radical groups of tyrosine tryptophan and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum tungsten blue The bound reagent changes color from yellow to blue This binding is compared with that obtained with a standard protein at 750 nm this is usually BSA bovine serum albumin Note Detailed protocols are customarily supplied with these assay kits and must be closely followed to ensure that accurate results are obtained MEASUREMENTS PROTOCOL Note It is not recommended to use the NanoVolume application for Protein Assays For best results use the cuvette mode NP80 and C40 models only for the Protein Assays 1 Select protein assays icon from home screen To change between NanoVolume and cuvette application use the Change to Cuvette NanoVolume button below the parameter area NP80 C40 only 90 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 2 Select protein assay type from drop down of the parameter
39. achloride e Chloroform e Toluene e Phosphate containing buffers e Ether e PBS pH 4 10 e HEPES e Citrate e Hexane e Borate e Chloride salts MES e Acids gt pH 2 e Methanol e Bases pH 10 Note Highly concentrated acids and bases are not recommended It is recommended to wipe the sample surface with a lint free laboratory wipe immediately upon completion of each measurement For more information about compatibility of specific solvents not listed above please contact the Implen support team Support implen de to check the compatibility 32 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD BATTERY OPERATION The optional battery operation is only available for NP80 Touch N60 Touch and C40 Touch models A full battery pack can be operated for approximately 8 hours within normal usage i e 20 30 measurements per hour When the battery gets low there will be a first warning message Battery low and a bleep is sounded At this status it is possible to use the instrument under standard conditions at least for 1 hour It is recommended to charge the battery at this status If the battery is not charged there will be a second warning message when the battery is empty and several beep sounds before the instrument is automatically shutting down within 10 to 15 seconds Note Unsaved data can be found in the autosave folder in Stored Methods If the NanoPhotometer is shutting down during a measur
40. acids the concentration of the nucleic acid is calculated using a modified form of the Beer Lambert equation For these calculations the instrument considers the absorption maximum of the dye and a certain dye specific correction factor at 260 nm see 36 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Table 2 The concentration of a dye labeled nucleic acid is calculated with or without background dye correction as follows With background and with dye correction C Ace A320 cfaye Anae A320 Enc D With background and without dye correction A260 A320 Enuc Without background and with dye correction C Cfaye Enuc Without background and without dye correction C A260 Enuc D C Concentration in ng ul 260 10 A320 Absorbance at 320 nm 10 mm path Amax dye Absorbance value at the absorbance maximum of the dye 10 mm path Bons Extinction coefficient nucleic acid factor in ng cm ul Cfaye Dye dependent correction factor at 260 nm D Manual dilution factor Dye Concentration For dye labeled nucleic acids the concentration of the dye is calculated using a modified form of the Beer Lambert equation For these calculations the instrument considers the absorption maximum of the dye the dye specific extinction coefficient see 3 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1
41. ample button Upon completion of measurement remove the lid clean the surfaces and apply the next sample 30 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 SAMPLE HANDLING TIPS Nanovolume Methods The NanoPhotometer includes an integrated vortexer N60 NP80 only to assure sample homogeneity It is recommended to vortex every sample right before the measurement The sample window is illuminated NP80 N60 N50 only with a low energy red light to assist with accurate sample application The red light is switched off once the sample arm is closed It is possible to disable the illumination feature in preferences of the NPOS The minimum volume that can be used for nanovolume samples is 0 3 ul starting for NP80 N60 N50 dsDNA 420 ng ul and BSA 12 6 mg ml and for submicroliter cell in the NP40 dsDNA gt 25 ng ul and BSA gt 0 7 mg ml For automatic path length setting at least 1ul is needed NP80 N60 N50 only maximum volume that can be used for nanovolume samples is 2 0 ul NP80 N60 N50 and 5 ul for the submicroliter cell with lid 5 The sample can be fully recovered after measurement with a pipette if desired Note Minimal cross contamination cannot be avoided on a molecular level Proper cleaning is important to ensure accurate measurements In most cases a dry lint free laboratory wipe is sufficient to clean the sample quartz surfaces In the case of highly concentrated samples or certain proteins the r
42. and Illumination Sample Window DISPLAY The brightness of the built in screen can be adjusted under display Note Display preferences are only available for instruments with integrated touchscreen ABOUT In about the following information about the NanoPhotometer are shown NanoPhotometer model serial number IP address hardware version firmware version and software WiFi ACCESS POINT Toggle Switch to en disable the WiFi access point ILLUMINATION SAMPLE WINDOW Toggle switch to switch on off the illumination of the sample window for NP80 N60 N50 only Note Only available for NP80 N60 N50 models ENABLE NANOVOLUME Toggle switch to enable the submicroliter cell option in the NanoPhotometer C40 only DYES There is a list of available dye labels for both nucleic acid dye and protein dye methods Each dye has either an icon in front of the dye name indicating that the dye is locked and cannot be changed or deleted If the dye has a delete symbol there is an option to delete the dye which is not locked or pre programmed into the NanoPhotometer Selecting a dye name opens a new screen with the dye information dye name absorbance maximum dye dye dependent extinction coefficient y M and dye dependent correction factor as well as the option to show the dye in the drop down of the application Nucleic Acid or Protein UV 68 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 4 Pre
43. and that the power connection is not interrupted during the update CONTACT IMPLEN There is an option to contact Implen for instruments connected to the internet For any support issues and questions please contact the Implen team directly Europe Asia South Pacific Middle East and Africa Implen GmbH Schatzbogen 52 81829 M nchen Germany Phone 49 89 72637180 Fax 49 89 726371854 Email support implen de www implen de North and South America Implen Inc Unit 104 31194 La Baya Drive Westlake Village CA 91362 USA Phone 1 818 748 6400 Telefax 1 818 449 0416 Email support implen com Website www implen com 71 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 8 MAINTENANCE MAINTENANCE FREE TECHNOLOGY The NanoPhotometer technology is maintenance free Regular maintenance and calibration is not necessary For facilities that are working according to national as well as international guidelines and standards including Good Laboratory Practice GLP Good Manufacturing Practice GMP or ISO9000 9004 the proper performance of the spectrophotometer has to be tested and proven on a regular basis with individually set intervals Implen provides certified NanoPhotometer secondary standards as an optional accessory These NanoPhotometer Didymium glass filters and standard solution sets are suitable for the control and documentation of the wavelength accuracy and the photometric accuracy of your system
44. any shortages are discovered inform your supplier immediately Inspect the instrument for any signs of damage caused in transit If any damage is discovered inform your supplier immediately Ensure your proposed installation site conforms to the environmental conditions for safe operation indoor use or dry environment Note Do not expose your NanoPhotometer near liquids rain moisture or dusty environments Temperature range 10 40 C If the cuvette heating is used the range 15 10 30 C If the instrument is subjected to extreme temperature changes it may be necessary to allow the instrument to equilibrate and to turn the instrument off and then on again once thermal equilibrium has been established 2 3 hours Maximum relative humidity non condensing of 80 and up to 31 C decreasing linearly to 50 at 40 C The instrument must be placed on a stable level surface that can support 4 5 kg and so that air can circulate freely around the instrument Ensure while powered on that no materials reduce air circulation The equipment should be positioned such that in the event of an emergency the main plug can be easily located and removed Carry the instrument always by holding the main corpus of the instrument and not e g on the optional attached display or NanoVolume pedestal The equipment must be connected to power with the 60W power supply cord supplied by Implen 90W with battery pack It can be used on 90 250 V 5
45. arameter for the measurement Note Choose the dilution lid depending on your sample concentration Lid dsDNA ng ul BSA mg ml absorbance range _ 20 05 75 Ee 22 EC 15 25 mE 0 70 108 0 50 150 n optional 50 7 500 1 45 217 1 00 150 250 optional 125 18 750 3 63 543 2 50 375 4 Ensure that the sample window and the mirror in the dilution lid are clean 5 Mix the sample very well to achieve a homogenous sample 6 Pipette the appropriate sample volume onto the center of the measuring window The required volumes are also shown in the parameter area of the software 03 2 ul 100 optional 0 3 2 ul 1 100 250 optional 0 3 2 yl 1 250 Note Do not overfill the well 7 Ensure that for the measurements the lid fits exactly onto the positioning supports mounted to the body of the cell and initiate a blank measurement with the blank button Note UV exposure Do only initiate a measurement when the lid is closed 8 Clean the measurement window and mirror in the lid with a slightly wet lint free tissue Use water 7096 EtOH or isopropanol Note Do not use aggressive solvents such as strong acids or bases or organic solvents at any time If unsure please contact support implen de for detailed information about your specific reagent solvent 9 Apply the appropriate amount of sample solution onto the measurement window and initiate the sample measurement with the s
46. blank ddH20 or buffer to the illuminated sample window for the reference measurement and select blank to initiate the reading Note Use always a minimum of 1 ul for any blank measurement Note he illumination of the sample window can be switched off in the preferences 9 Use a lint free laboratory wipe to clean both the sample window and mirror prior to applying the next sample Note It could be helpful to apply the blank a second time and read itas a sample to ensure a proper blank 10 Apply sample to the sample window and press the sample button to initiate the measurement CALCULATIONS The absorbance ratio is calculated from the two path lengths specified by the user in the parameters des Au Ao Absorbance Ratio Absorbance 1 corresponding absorbance value 1 selected normalized to 10 mm path gt Absorbance 2 corresponding absorbance value 2 selected normalized to 10 mm path x 61 MORE APPS CONCENTRATION NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD METHOD OVERVIEW In this mode concentration can be calculated for a sample by determining the absorbance at a specific wavelength relative to a reference The concentration is then obtained by multiplying the measured absorbance by a specific factor This factor may be known in advance and entered by the user or it may be calculated by the instrument by measuring a set of standard standard curve method with known concentrations to
47. create a standard curve MEASUREMENTS PROTOCOL x 140 0 07 mm SE Heat 37 C ISS Wavelength 4 1 Select the More Apps icon the Home screen and then the Concentration icon from the More Apps screen For NanoVolume application Select the dilution depending on the sample concentration Note There is no automatic path length setting in this method Select either a virtual dilution of 15 path length 0 67 mm or of 140 path length 0 07 mm For cuvette application Select the path length depending on the used cuvette Options are 1 2mm 5 and 10 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown If it is desired to heat the sample to 37 C use the toggle switch to turn on cell holder heater When the cuvette holder has 37 C the color changes to green Note Only available for cuvette applications NP80 and C40 Default wavelength is 260 nm but can be changed depending on the application Enter a factor for concentration calculation Baseline correction 1 off as the default and can be enabled by selecting the different values for the correction in the drop down menu 377 nm 604 nm 770 nm N A N50 and 823 nm N A N50 Smoothing
48. e Software Installation NanoPhotometer Power Adapter Use the power adapter to plug in the NanoPhotometer for use and or charge the NanoPhotometer battery touch and mobile models with optional integrated battery Note Use only the power adapter supplied with your instrument or a replacement part from the manufacturer or your supplier Dust Cover OPTIONAL ACCESSORIES Didymium Glass Filter C40 NP80 The certified didymium glass filter is used as a control of wavelength and photometric accuracy for the NanoPhotometer cuvette applications 12 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Submicroliter Cell C40 only The submicroliter cell provides the option of upgrading the NanoPhotometer C40 to a device that handles nanovolume samples as well as cuvette samples The sample volume range of the submicroliter ranges from minimum of 0 3 ul to 5 0 ul with the path length options including 0 04 mm 0 1 mm 0 2 mm 1 mm and 2 mm which correspond to virtual dilution factors of 1 250 1 100 1 50 1 10 and 1 5 respectively MELEN DiluCell C40 NP80 only The DiluCell is a disposable cuvette with shortened path length for virtual dilution of cuvette based samples Due to the reduced path length the DiluCell provides an automatic dilution without the need of a physical dilution of higher concentration samples There are two different sizes of DiluCells avai
49. e on tablets and smartphones not on the NanoPhotometer and computer versions of the software W STORED METHODS The stored methods icon opens the directories of folders containing methods stored by the user sz Stored Methods NanoPhotometer USB Flash Drive On the left side of the screen all available directories are shown NanoPhotometer and or USB flash drive To open a folder click on the gt icon for selecting the folder click on the folder name 66 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 Gi New folders can be created by clicking Folders can be deleted renamed moved or copied to by clicking on the icon On the right side of the screen all saved methods of the selected folder are shown and can be opened by a long or double click It is also possible to delete rename move or copy methods by clicking on the 4 icon 67 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 5 PREFERENCES System preferences can be set by selecting preferences RE on the home screen The preferences menu includes general and dyes The menu item which is selected will have its preference options listed in the window to the right Note Preferences are not available for smart phone 7 screen versions GENERAL Selecting General in the Preferences menu opens a window to the right of the preferences menu with the following options Display built in display only About WiFi Access Point
50. e to standard cuvette and all in one solutions The N50 N60 and NP80 spectrophotometers measure sample volumes ranging from 0 3 2 ul The NP80 model also has the capability to measure samples using a standard cuvette The C40 is a mobile standard cuvette solution with the option to upgrade to accommodate nanovolume measuremenis The NanoPhotometer runs on a Linux based operating system NPOS that is designed for the use of pre programmed and custom applications with a high degree of flexibility and processing power Sample Compression Technology provides easy sample handling which is independent of surface tension This technology squeezes the sample between two quartz surfaces allowing for unmatched precision and accuracy without the need for dilutions Combined with our True Path Technology the system offers lifetime accuracy and precision without the need for maintenance or recalibration Note is recommended to use a properly calibrated pipette with high quality tips to ensure delivery of appropriate sample volumes for nanovolume sample applications NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 NANOPHOTOMETER NP80 7 Color Touch Screen Illuminated Sample Window ht a E Temperature Controlled Cuvette Holder Power Indicator Low Vibration Vortexer Front USB Port Item Number Touchscreen Battery Pack NP80 NP80 Touch NP80 Mobile NanoPhotometer NP80 N60 N50 C40 User Guide Vers
51. ecommended procedure for cleaning is to use a slightly wet lint free laboratory wipe with water or 70 EtOH depending on sample type to thoroughly clean the sample surface It is mandatory that the metal contact face around the measurement window and the mirror is clean Cuvette Methods The cuvette holder NP80 C40 only is compatible with standard 10 mm path length quartz glass and plastic cuvettes with an optical height of 8 5 mm is also possible to use cuvettes with 5 mm 2 mm or 1 mm path lengths but there may be an adapter necessary Please ask your cuvette supplier for suitable adapter 31 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD e he minimum volume for accurate measurements depends on the cuvette type used it is necessary for the light path to pass through the sample for accurate measurements Note The cuvette holder is not removable Do not pour any cleaning solution into the cuvette holder as larger amounts of liquids can get into the instrument and cause damage Solvent Compatibility N50 N60 NP80 C40 with submicroliter cell Most solvents typically used in life science laboratories are compatible with the NanoPhotometer nanovolume sample surfaces The following solvents are compatible for use with the NanoPhotometer models N50 N60 and NP80 at room temperature e Acetone x 5 e Methylenchlorid e Acetonitrile e MOPS e Benzene e Phenol lt 1 e N propanol Carbon tetr
52. ement process the data of this measurement are lost The battery pack is designed for 800 full charging cycles Afterwards the capacity and operation time of the battery may vary A fully charged battery pack will self discharge switched off NanoPhotometer over a time of 10 14 days After this time recharging of the battery pack is necessary 33 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 4 NANOPHOTOMETER APPLICATIONS The NanoPhotometer comes with pre programmed applications as well as the ability to create custom applications Any application method can be selected by tapping the icon once or clicking on the icon computer based software Note To select a method select the corresponding icon Selection of nanovolume or cuvette mode is within each method possible The Change to Cuvette or Change to NanoVolume button is located at the bottom of the parameter area NUCLEIC ACIDS METHOD OVERVIEW Nucleic acids in solution absorb light with a peak in the ultraviolet region of 260 nm For determination of nucleic acid concentration in solution the absorbance at wavelength 260 nm is used along with the Beer Lambert law In addition to calculating concentrations of nucleic acids absorbance measurements are also useful for estimating purity of nucleic acids by calculating the 260 280 nm and 260 230 nm ratios Further it is possible to determine the degree of labeling of nucleic acids with probes includin
53. erences Use a lint free laboratory wipe to clean both the sample window and mirror prior to applying the next sample Note It could be helpful to apply the blank a second time and read it as a sample to ensure a proper blank Apply sample to the sample window and press the sample button to initiate the measurement No calculations necessary values are reported based on 10 mm path length In the results are the five prominent peaks shown with wavelength and absorbance value For cuvette measurements it is possible to change to the Transmittance mode If a peak of interest is not shown in the results the peak can be added by clicking on the graph and added to the results by tapping on the Add Peak button in the pop up window Sample Name Wavelength Absorbance Sample22 0 771 259 59 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 MORE APPS ABSORBANCE METHOD OVERVIEW In this mode it is possible to determine simple absorbance ratios for a given sample by measuring the absorbance at two wavelengths specified in the parameters of the method relative to a blank MEASUREMENT PROTOCOL 1 Select the More Apps icon from the Home screen and then the Absorbance Ratio icon from the More Apps screen 2 For NanoVolume application Select the dilution depending on the sample concentration Note There is no automatic path length setting in this method Select either a virtual diluti
54. ferences PREFERENCES lt el Dyes Dye Absorbance 550 Maximum Dye nm Coefficient Dye dependent Correction Factor 260 nm CFlabel Note It is not possible to delete a dye of the factory list custom dyes can be deleted if they are not locked is possible to add a new to the list by selecting the button to add a new dye A window will open where it is possible to enter the dye name dye absorbance maximum nm dye dependent extinction coefficient y and dye dependent correction factor There is toggle switch available to lock the dye to prevent deleting a dye from the dye list accidentally 6 TROUBLESHOOTING SELF CALIBRATION TEST The NanoPhotometer self calibration test is performed automatically every time the instrument is powered on If the instrument passes the self calibration test the home screen is shown If the instrument does not pass the test a window with a message will appear along with the message explaining the reason for the failed test along with the recommended solution If the ok button is selected the window is closed and the home screen will be shown with an error message at the top The error message is also shown on top of all other screen and all performed measurements including the saved files of those measurements If the self calibration test fails please contact the Implen Support Team 7 ASSISTANCE The Assistance me
55. g fluorescent dyes MEASUREMENTS PROTOCOL 1 Select the Nucleic Acids icon on the home screen To change between NanoVolume and cuvette application NP80 C40 only use the Change to Cuvette NanoVolume button below the parameter area 2 Select the nucleic acid type in the drop down of the parameter area Options are dsDNA ssDNA RNA miRNA miRNA Sequence Oligo Oligo Sequence and Custom see Table 1 The miRNA sequence and Oligo sequence options allow having the sequence entered and the extinction coefficient will be automatically calculated ED Custom is set to 50 as a default and allows the extinction coefficient to be Oligo manually entered from the range of 15 150 up to three decimal points Oligo Seq Custom 3 For NanoVolume application select the volume of sample to be applied Note 1 2 ul default automatic path length change 0 3 measures only the 0 07 mm path length for samples with concentrations gt 420 ng ul dsDNA 34 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Background 5 Add Dye Label D 6 G9 Select Dye v Dye Correction Wi Manual 8 For cuvette application Select the path length depending on the used cuvette Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for
56. gent solvent 7 Raise the sample arm and pipette the appropriate amount of sample solution onto the illuminated sample window 1 2 ul for automatic path length change or 0 3 ul for dsDNA gt 420 ng ul BSA 12 6 mg ml Upon completion of measurement raise sample arm clean the surfaces and apply the next sample Note The sample window must be clean and residual fluff from any cleaning wipe must be removed for optimum performance CUVETTE MEASUREMENT Basics C40 NP80 The NanoPhotometer NP80 and C40 only is compatible with standard cuvettes having an 8 5 mm center height The light path is indicated with a red status LED arrow for the NP80 model and with two white arrows for the 40 model Note For the NP80 the cuvette compartment needs to be activated by the Change to Cuvette button in the parameter area Once the cuvette option is activated the sample compartment door will be opened automatically and a red arrow above the cuvette compartment will appear The arrow indicates the light path 1 Select a method depending on your sample and set the parameter for the measurement 2 Open the cuvette cover by clicking the Change to Cuvette button below the parameter area NP80 28 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Nucleic Acids 686 45 250 400 A260 A280 1448 Wavelength 65506220 FI Concentrat Units A260 A260 A280 A26O A230 3 Add a blank solution to
57. gle switch ve EY Added Dyes can be deleted by tapping on the delete icon 09 Note If the used dye is not available in the drop down list please go to preferences and add a custom dye to the dye list Manual 7 Option to set calculate a dilution factor for manual diluted samples Dilution BLANK 8 Apply the blank ddH20 or buffer to the illuminated sample window for the reference measurement and select blank to initiate the reading Note Use always a minimum of 1 ul for any blank measurement Note The illumination of the sample window can be switched off in the preferences 9 Usea lint free laboratory wipe to clean both the sample window and mirror prior to applying the next sample Note It could be helpful to apply the blank a second time and read it as a sample to ensure a proper blank 10 Apply sample to the sample window and press the sample button to initiate the measurement CALCULATIONS Protein UV280 Concentration The protein concentration in the Protein UV method is calculated with the absorbance value of the sample at 280 nm along with the extinction coefficient defined by the user The protein concentration is calculated with or without background correction as follows 43 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 With background correction C A280 A320 D Without background correction C A280 C Concentration ng ul A280 Absorbance at 280 nm
58. gnetic compatibility generic emission standard electrical equipment for measurement control and laboratory use Signed T VW Ba SQL MA Dr Thomas Sahiri Managing Director Implen GmbH NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Contents 1 NANOPHOTOMETER 6 OVERVIEW 6 NANOPEHOTOMETERH 92922020922 00202 7 N60 mm 8 NANOPHOTOMETER LN 9 NANOPHOTOMEIEH 10 INSTRUMENT REAR PANEL 6 5 11 INSTRUMENT BOTTOM VIEW 80 60 50 40 nauta unn uaa RER E EEN 11 PAC CES e TEE 12 STANDARD 12 OPTIONAL ACCESSORIES PIED II UE DEP E EDI e I
59. his case the entire measurement session is saved Save as The save as button allows the user to specify which type of file to save the data as The options of file types include Excel and Implen Document Source IDS which can be saved to NanoPhotometer Control Device and on USB flash drive Note Control Device is only available on smart phones and tablets 25 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Implen Document Source File Implen has a custom file format referred to as being an Implen Document Source IDS file This is a hardcopy file to which no changes can be made This file type contains all information including raw data results values and parameters The saved file shows only the selected information at the time the file was saved IDS files can only be opened with the Implen software The data are opened in the measurement Excel File Data from the measurements can also be saved in an Excel file format This file format is compatible with Microsoft Excel software versions 2007 2010 2013 and can be edited This file type contains all information including raw data results and parameters The saved file contains only the selected information at the time the file was saved Storage oelection option to choose the device the data should be saved to The options include NanoPhotometer USB flash drive if connected and Control Device for smart phones and tablets only If Control Device is selected
60. ion 1 1 0 NANOPHOTOMETER N60 7 Color Touch Screen Illuminated Sample Window Power Indicator Low Vibration Vortexer Front USB Port Item Number Touchscreen Battery Pack N60 N60 Touch N60 Mobile NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 Qu NANOPHOTOMETER N50 7 Color Touch Screen Illuminated Sample Window Power Indicator Front USB Port Item Number Touchscreen Battery Pack N50 N50 Touch NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 NANOPHOTOMETER C40 7 Color Touch Screen Temperature Controlled Cuvette Holder Power Indicator Front USB Port Item Number Touchscreen Battery Pack C40 C40 Touch C40 Mobile 10 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD INSTRUMENT REAR PANEL NP80 N60 N50 C40 Slot for Power Supply USB B HDMI USB A Ethernet Power On Off Kensington lock INSTRUMENT BOTTOM VIEW NP80 N60 N50 C40 IMPLEN GMBH Model NanoPhotometer NP80 Mobile SGS Serial No M80701 er Input 19V 47 Model name device serial number and ID are located on the identification plate on the bottom of the instrument 11 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD ACCESSORIES STANDARD ACCESSORIES Connecting Cable The USB cable provided makes it possible to connect the NanoPhotometer to a computer to control the instrument from the computer please se
61. lable DC 10 and DC 20 which allow an automatic 1 10 and 1 20 dilution of the sample respectively Bypassing manual sample dilutions reduces dilution errors and cross contamination making DiluCell ideal for GLP Combined with small sample volume requirements and bubble free filling the Dilucell allows for convenient spectrophotometric analysis from 340 950 nm Field Kit Implen aluminium roller case for safe transportation designed to fit in most airplane overhead bins special compartments for all necessary accessories cleaning tools and samples Note Delivered w o NanoPhotometer and accessories Note Only operate the NanoPhotometer in the roller case when cover is open Ensure that air circulation is possible Switch the NanoPhotometer always off for transportation 13 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD CONNECTIVITY WiFi The NanoPhotometer has a WiFi hotspot capability This provides the option to control the NanoPhotometer by other WiFi devices like smart phones or tablets SSID NanoPhotometer serial number password Implenuser USB A There is a USB A port on the front and rear panel of the NanoPhotometer which is compatible with standard portable USB 2 0 storage devices for direct data transfer in a variety of formats including Excel It is also possible to connect a mouse or keyboard directly to the NanoPhotometer Note We recommend to use FAT FATS32 formatted 2 0 USB flash drives
62. ly tryptophan and tyrosine as well as cysteine oxidized cysteine residues in a disulphide bond The aromatic amino acid residues in a protein containing tryptophan and tyrosine exhibit strong intrinsic absorbance at 280 nm with a lesser contribution by phenylalanine Therefore it is the aromatic amino acid residues which dictate the extinction coefficient at 280 nm for a protein The most straightforward method to determine concentration of a purified homogenous protein with a known extinction coefficient 15 by direct measurement of UV280 provided as long as the protein contains no prosthetic groups with strong absorption in the same region However for unknown proteins including homogenous protein mixtures it is possible to make direct Asso measurements using a composite value derived from comparison of many proteins although this will only provide an approximate but close estimate of the true protein concentration The NanoPhotometer determines protein concentration by performing calculations based on specific values either pre programmed in the instrument or entered manually by the user Extinction coefficient values at 280 nm vary greatly for different proteins due to their particular aromatic amino acid content Fixed values are pre programmed in the software for certain proteins seeTable 3 However if the protein of interest is not included in the pre 41 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD
63. mart phone only Next Confirm Confirms parameter and opens the next screen smart phone only Delete Deletes added functions in parameter empties input windows simis i Delete Data Opens a delete dialog pop up mail Data pens an email dialog pop up computer and tablet versions only VEA Email Dat ter and tabl Save Data Opens a save dialog pop up Adds a new folder to the directory 1 Opens dialog pop up with several action options including delete AN Manage Data rename or import folders files data as well as copying or move folders files data to defined directories Store Method Opens a dialog pop up with the possibility to store the actual method parameter to a custom method AK Full Scale Restores graph to original size without zoom X Cancel Returns to the previous screen without implementing any changes BUTTONS Prior to any sample measurement at the start of a new method a blank measurement of either water or the buffer of the samples is required to give the NanoPhotometer a reference of what zero should be It is recommended to re apply the blank and measure it as a sample to ensure the graph of the blank spectra is a flat line Note Use always a minimum of 1 ul for any blank measurement 22 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD To initiate the spectral scan of the sample press the sample button The data will be temporarily stored until the method is exited
64. n 1 1 0 QD 00600 METHOD OVERVIEW The growth of bacteria in liquid culture media is commonly monitored by measuring the optical density at 600 nm OD600 in small samples taken from the cultures OD600 measurements are typically used to determine the stage of growth of the bacterial culture thereby ensuring that cells are harvested at an optimum point that corresponds to an appropriate density of live cells Growth of bacterial cells typically progresses through a series of consecutive phases including lag log stationary and decline see Figure 1 In general cells should be harvested towards the end of the log phase using the optical density of the samples to determine when this point has been reached Since optical density in the case of OD600 measurements results from light scattering rather than light absorption this value varies depending on the type of bacterial cells in the culture in terms of size and shape Cells are routinely grown until the absorbance at 600 nm known as OD 600 reaches approximately 0 4 prior to induction or harvesting A linear relationship exists between cell number density and OD 600 up to an absorbance value of 0 6 approximately As mentioned above for turbid samples such as cell cultures the absorbance measured is due to light scattering and not the result of molecular absorption Since the extent of scattering is affected by the optics of the system distance between the cell holder and instrument exit
65. nce ratio standard curve and custom applications 55 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 MORE APPS WAVELENGTH METHOD OVERVIEW In the wavelength application it is possible to measure simple absorbance A and 96 transmittance Trans only in cuvette mode of a sample at specific wavelengths It is possible to add up to 20 different wavelengths MEASUREMENT PROTOCOL 1 Select the More Apps icon from the Home screen and then the Wavelength icon from the More Apps screen 2 For NanoVolume application Select the dilution depending on the sample concentration Note There is no automatic path length setting in this method Select either a virtual dilution of 15 path length 0 67 mm or of 140 path length 0 07 mm imm For cuvette application Select the path length depending on the used cuvette Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown 3 If itis desired to heat the sample to 37 use the toggle switch to turn on cell holder heater When the cuvette holder has 37 the color changes to green Note Only available for cuvette applications NP80 and C40 A 260 nm 4 Enter desired wavelength X to be mea
66. nt areas on the screen Default screen for computer show all areas for the built in screen and tablet version the table is hidden A 2 Nucleic Acids Hm EN dsDNA v 50 Parameter CONNU Volume 1 20 v Concentration N A Absorbance 10 mm Background WW Q Results AddDyeLabel 0 e ir m 400 2 2 Manual Dilution KS Overlay Edit Name Concentrat Sample4 ES Table Change to Cuvette enter sample name SAMPLE 23 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Note There is no tab bar available for smart phone versions The parameters results and graph screens are shown full screen Parameters need to be confirmed gt to get to the measurement screen It is possible to toggle between the results and graph area by sliding left and right There is no table area available on smart phones Parameter area In the parameter area it is possible to define all necessary parameters for a measurement as well as turn on cuvette mode and initiate cuvette heating The default measurement screen includes the parameter area as open It is automatically hidden when starting either a blank sample measurement by clicking on the Blank or Sample button It is also possible to hide the parameter area by tapping the parameter tab in the vertical side tab bar Results area The results area shows the method specific result
67. nu includes support report a problem only available for tablet and computer versions software maintenance and legal as functions to help with any technical issues or questions that may arise with the NanoPhotometer The menu list item selected on the left side assistance menu will appear in the window to the right Note Assistance function is not available for smart phone lt 7 screen versions 69 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD SUPPORT Selecting support on the left side assistance menu will show the available options for contacting Implen ASSISTANCE FOR ANY SUPPORT ISSUES AND QUESTIONS PLEASE CONTACT THE IMPLEN TEAM Support EUROPE ASIA SOUTH PACIFIC MIDDLE EAST AND AFRICA Report Problem Software Maintenance 31829 M ncher support implen de www implen de Legal NORTH AND SOUTH AMERICA REPORT PROBLEM The function to report a problem is available on computer and tablet versions only When report a problem is highlighted in the left side assistance menu the right side will have a form to fill out with information including first name last name phone number email and country A drop down menu provides the option to select the problem type and includes the following selections error message software firmware measurements and other It is also possible to enter a question or comment at the end of the form Once the form is completed and the send button is selec
68. of the samples DNA and preparations have expected 260 280 ratios of gt 1 8 and gt 2 0 respectively An elevated absorbance at 230 nm can indicate the presence of impurities as well 230 nm is near the absorbance maximum of peptide bonds and also indicates buffer contamination since TRIS EDTA and other buffer salts absorb at 230 When measuring samples the 260 230 ratio should be gt 2 0 a ratio lower than 40 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD this is generally indicative of contamination with guanidinium thiocyanate a reagent commonly used in RNA purification and which absorbs over the 230 260 nm range The ratios are calculated with or without background correction according to if the background correction is activated during the measurements or not as follows Without background correction 260 280 ratio 2 A280 260 230 ratio Ces 230 With background correction 260 280 ratio 4290 s2o A280 A320 260 230 ratio Ze Zen 230 20 PROTEIN UV METHOD OVERVIEW The Protein UV method exploits the inherent absorbance of proteins at 280 nm in combination with the Beer Lambert Law where each protein is characterized by a protein specific extinction coefficient which can be used to determine total protein concentration of a solution The intrinsic absorbance of proteins is due to the presence of aromatic amino acids in their structure primari
69. on of 15 path length 0 67 mm or of 140 path length 0 07 mm For cuvette application Select the path length depending on the used cuvette Options are 1 mm 2 mm 5 mm and 10 mm For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown 3 If itis desired to heat the sample to 37 use the toggle switch to turn on cell holder heater When the cuvette holder has 37 the color changes to green Note Only available for cuvette applications NP80 and C40 4 Enter desired wavelengths X 1 1 and X 1 2 for ratio calculation It is possible to measure up to 20 absorbance ratios simultaneously More wavelength for ratio calculation can be added by selecting the Add Ratio button The added ratios can be deleted with 60 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 5 Baseline correction is off as the default and can be enabled by selecting the different values for the correction in the drop down menu 377 nm 604 nm 770 nm N A N50 and 823 nm N A N50 Smoothing Of 6 Option to smooth the graph with different boxcars off 1 boxcar 11 default 2 boxcar 21 and 3 boxcar 61 7 Option to set calculate a dilution factor for manual diluted samples 8 Apply the
70. ons may have not the full functionality FIRST STEPS AND CONFIGURATION WIZARD otarting the Implen NPOS the first time an Implen configuration wizard is shown Please confirm the End User License Agreement EULA and select the country in which the NanoPhotometer is used and confirm 19 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 3 NANOPHOTOMETER BASICS The NanoPhotometer product line offers a complete solution for Nanovolume NP80 N60 N50 C40 and standard cuvette C40 NP80 applications With the NanoPhotometer nanovolume applications the sample volume ranges from a minimum of 0 3 ul to a maximum sample volume of 2 0 ul 5 ul for using the submicroliter cell C40 Standard cuvette applications can be performed with 10 mm 5mm 2 mm and 1 mm path length quartz glass or plastic cuvettes with a center height of 8 5 mm APPLICATIONS OVERVIEW The NanoPhotometer comes with pre programmed applications as well as the ability to create custom applications To select a method click tap on the corresponding icon and the method will open immediately Ve Stored Methods Method Icons Description Concentration purity and dye incorporation for DNA RNA Oligo and other nucleic acids Protein UV determination at 280 nm or in a range of 200 330 nm purity and dye incorporation Time vs Absorbance readings BCA 562 nm Bradford 595 nm Lowry 750 nm and Biuret Assays 546 nm OD600 SEN cell densit
71. otential danger exists or particular caution is required direct current Do not open the instrument as this can expose the operator to electrical power UV light and delicate fiber optics If the submicroliter cell is used with the NanoPhotometer C40 make sure that the lid is placed on the measurement head prior to the measurement WARNING UV exposure when measuring without lid Do not use damaged power cords accessories and other peripherals with your NanoPhotometer Do not expose the NanoPhotometer to strong magnetic or electrical fields Do not leave your NanoPhotometer on your lap or near any part of your body to prevent discomfort or injury from heat exposure Do not place objects on top of your NanoPhotometer The NanoPhotometer with battery pack mobile version has to be switched off during transportation The on off button has to be protected from turning on itself caused by shock or vibration 16 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 IMPLEN a The symbol on the product or on the documents accompanying the product indicates that this appliance may not be treated as household waste Instead it shall be handed over to the applicable collection point for the recycling of electrical and electronic equipment Disposal must be carried out in accordance with local environmental regulations for waste disposal Unpacking and Positioning Check the contents of the package against the delivery note If
72. rument to the computer using the USB cable provided Note The Windows and Mac installation file is provided on the Implen USB flash drive which is included in the NanoPhotometer delivery and are available for free download in the download section of the Implen webpage Note If there is Avira installed on your computer it is recommended to switch off the browser safety This may interfere with the NPOS running on your computer INSTALLING NANOPHOTOMETER APP ON TABLET OR SMART PHONE The NanoPhotometer App can be installed as an application on tablets and smart phones with compatible Android and iOS operating systems The NanoPhotometer App is available for free download in the app store Apple Store and Google Store 1 Download and install the NanoPhotometer App from the app store 2 Use the WiFi to connect the NanoPhotometer to the tablet or smart phone SSID Serial number password Implenuser 3 Open the NanoPhotometer App 4 When connected via WiFi the NanoPhotometer will recognize the tablet as a remote control device and measurements can be initiated from the tablet or smart phone 5 Measurement results will be shown to the tablet or smart phone once taken Note In order to install the NanoPhotometer App on a tablet or smart phone the device must have an established internet connection to access the app store for app download Note The version of the app and the software of the NanoPhotometer should be the same Different versi
73. s of the latest measurement including concentration absorbances and relevant ratios from the last sample measured It is also possible to change the units of the calculated concentrations in the results area with a drop down selection menu Note Selection of one or more than one line is selected in the table no data are shown in the result view Table area The table area collects defined results for all samples completed during the active method The first column of the table indicates whether the measurement is saved or not saved blank field Graph area The graph area shows a chart with the graph of the actual measurement or the selected line s in the table There is an overlay toggle switch on the left bottom of the graph area If the overlay option is enabled the graphs of the measurements will be automatically overlaid To change the overlaid graphs select deselect the samples in the table Note It is only possible to overlay up to 30 graphs in a chart If more than 30 data are selected a message will appear that says Too many data selected Up to 30 data can be overlaid Please select the desired data to be displayed on the graph Note The overlay button is not available on the NanoPhotometer and smart phones only on tablets and computer versions It is possible to zoom in and out any area of the diagram x and y axis Undo the zoom by clicking the full scale icon Note Maximum zoom is 20 nm for
74. sorbance value at the absorbance maximum of the dye 10 mm path Extinction coefficient protein factor in 0 Cfaye Dye dependent correction factor at 280 nm D Dilution factor Dye Concentration For dye labeled proteins the concentration of the dye is calculated using a modified form of the Beer Lambert equation For these calculations the instrument considers the absorption maximum of the dye and a dye specific extinction coefficient see 46 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Table 4 The dye concentration is calculated with or without background correction for are as follows With background correction 20 C Eaye 10 Without background correction _ 5 Eaye 10 Concentration Amax dye Absorbance at the max dye absorbance value 10 mm path A320 Absorbance at 320 nm 10 mm path Extinction coefficient of dye in Mem D Dilution factor Degree of Labeling DOL DOL is the degree of labeling based on the average number of dye molecules coupled to a protein molecule The degree of labeling can be determined from the absorption spectrum of the labeled antibody with or without background dye correction as follows With background and with dye correction DOL Anax dye A320 Aa Cfaye dye With background and w o dye correction
75. standards of known concentrations can be created and stored on the NanoPhotometer The standard curve can be used to quantify samples of the same type with unknown concentrations This application provides an extremely useful tool with which to integrate expedite and simplify the measurement and calculations 63 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD involved in determining the concentration of analytes in unknown samples If a zero concentration standard is required include it in the number of standards to be entered using a reagent blank and entering 0 00 for concentration MEASUREMENTS PROTOCOL 1 Select the More Apps icon from the Home screen and then the Standard Curve icon from the More Apps screen For NanoVolume application Select the dilution depending on the sample concentration Note There is no automatic path length setting in this method Select either a virtual dilution of 15 path length 0 67 or of 140 path length 0 07 For cuvette application Select the path length depending on the used cuvette For NanoVolume application with submicroliter cell C40 only Select Lid depending on your sample concentration see also Submicroliter Cell Measurements Basics C40 for concentration ranges of different lids Required sample volume for each lid is shown below the lid selection dropdown 3 If itis desired to heat the sample to 37 use the toggle switch to turn on cell holder heater
76. sured It is possible to measure up to 20 wavelengths simultaneously More wavelength A options are added by selecting the wavelength button The added wavelength can be deleted with E A3 280 09 5 Baseline correction is default off and be enabled by selecting different values for the correction in the drop down menu 377 nm 604 nm 770 nm N A N50 and 823 nm N A N50 56 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD Smoothing Of 6 Option to smooth the graph with different boxcars off 1 boxcar 11 default 2 boxcar 21 and 3 boxcar 61 Manual Dilution T 7 Option to set calculate a dilution factor for manual diluted samples 8 Apply the blank ddH20 or buffer to the illuminated sample window for the reference measurement and select blank to initiate the reading Note Use always a minimum of 1 ul for any blank measurement Note he illumination of the sample window can be switched off in the preferences 9 Usea lint free laboratory wipe to clean both the sample window and mirror prior to applying the next sample Note It could be helpful to apply the blank a second time and read it as a sample to ensure a proper blank 10 Apply sample to the sample window and press the sample button to initiate the measurement CALCULATIONS Absorbance Calculation Absorbance is formally defined as the decimal logarithm base 10 of the reciprocal of transmittance
77. tallation process ensure the device is compatible with the requirements for installation Compatible Control Devices Computer PC Windows 7 Windows 8 32 bit or 64 bit Mac OS X Tablets iPad 2 iOS6 Android Quadcore 1 2 GHz with 1 GB RAM Android version 2 2 Smart phones iPhone 5 iOS6 Android Quadcore 1 2 GHz with 1 GB RAM Android version 2 2 Windows is a trademark of Microsoft Mac OS amp iOS are trademarks of Apple Android OS is a trademark of Google Linux is a trademark of Linus Torvalds Note There are two user interfaces of the software available one for built in touchscreen computer and tablets and one for smart phones INSTALLING SOFTWARE ON COMPUTER The NanoPhotometer software can be installed on compatible PC and Mac computer systems Various operating systems and PC hardware may cause the set up procedure to differ from that described here This process is given as guidance only it may need adaptation for other systems To prevent loss of data it is important to ensure that connections are stable and not lost during data transmissions and ensure that the system has enough time to save transferred sample data 18 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 1 install the NanoPhotometer software the installer should be logged with full administration rights If you have insufficient privileges installation may fail If in doubt consult your PC administrator 2 Connect the inst
78. ted a message will be sent directly to Implen and the appropriate support person will contact as soon as possible to provide further support Note The report a problem function is only available for computer and tablet versions SOFTWARE MAINTENANCE RESET There is an option to reset the instrument to factory settings By selecting the reset button a window will open that says Would you like to reset the NanoPhotometer Selecting the cancel button will close the window without changing the settings and selecting reset will open a window that will ask again Would you really like to reset the NanoPhotometer to factory settings If it is confirmed the factory settings will be restored Note All stored methods settings and data on the NanoPhotometer will be deleted if the reset option is executed 70 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD SOFTWARE UPDATE There is a button for software update It is needed to plug in a USB flash drive with the software installation file to the USB port The software update files are available on the Implen homepage www implen de Update procedure 1 Copy the NPOS bin file to a USB flash drive 2 Plug in the USB flash drive to the USB port of the NanoPhotometer 3 Go to Assistance Software Maintenance 4 Press the Update button The update will start and once finished automatically reboot the NanoPhotometer Note Make sure that the NanoPhotometer is connected to power
79. the measurement Note Once the sample measurement is initiated it is not possible to do changes to the standard curve CALCULATIONS Concentration is determined via the absorbance values provided by the standard curve based on the curve fit selection including the following options linear regression or zero regression CUSTOM APPS There is an option for designing customer specific Custom Apps which can be loaded to the NanoPhotometer For more information about designing custom applications to suit individual research needs please contact Implen directly for assistance STORED RESULTS The Stored Results icon opens the directories of folders containing files of results that have been previously saved 65 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD 2 Stored Results Control Device NanoPhotometer On the left side of the screen all available directories folders are shown NanoPhotometer Control Device and or USB flash drive To open a folder click on the gt icon for selecting the folder click on the folder name Folder can be created by tapping on Folders can be deleted renamed moved or copied to by clicking on the 4 icon On the right side of the screen all saved result files of the selected folder are shown and can be opened by a long or double click is also possible to delete rename move or copy files by clicking on the 4 icon Note Control device is only availabl
80. the x axis and 0 014 for the y axis 24 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD As legend option the sample name in the table is colored in the same color as the graph in the chart Clicking on the graph opens a pop up which shows the sample name wavelength and absorbance of the selected wavelength It is possible to show results of further graphs by changing the sample in the sample name drop down DATA PROCESSING DIALOGS Save Selecting the save data icon opens a full screen overlay dialog window with various save options Add folder Manage Data Name Concentrat Units A260 A260 A280 260 230 Cancel Select files for saving Samples Save selected Toggle switch save selected ee Save all Toggle switch save all Save as type Please select Select file format File Please enter file name Enter file name Storage NanoPhotometer Select folder for saving SAVE Save button Save selected This option saves the current selection of data of the table If the save selected toggle switch is green the save all toggle switch will automatically be deactivated Save all The save all button default setting allows the user to save all of the data from the current method If the save all toggle switch is green the save selected toggle switch will automatically be deactivated Note On the smart phone app there 15 no save selected save all toggle switch available instead in t
81. y at 600 nm or in a range of 200 900 20 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 QD p ISI Methods Collection of stored custom methods Results Archive of stored results Additional applications found on a secondary method screen Define one or multiple wavelength between Wavelength 200 950 nm N50 200 650 nm for absorbance measurements Define desired full scan range anywhere between Wavescan 200 950 nm N50 200 650 nm Define extinction coefficient for automatic Concentration concentration calculations Absorbance Define two wavelengths for a calculated absorbance Ratio ratio Standard Curve Create a standard curve at a defined wavelength Optional custom applications for personalized methods tailored to individual spectroscopy needs ei Custom Apps Note Protein Assays and OD600 applications the cuvette mode is recommended 21 NanoPhotometer NP80 N60 N50 C40 User Guide Version 1 1 0 ICONS Icon Name Action WiFi Hotspot WiFi hotsopt enabeld can be disabled in preferences 1 Assistance Opens the assistance page Battery Status Shows the actual battery status only shown with optional battery Preferences Opens the preferences page Screen Returns to the home screen with application icons for method selection Leave Method Returns to the previous application selection lt Returns to the previous page s

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