NanoDrop Spectrofluorometer V 2.5B User's Manual
Contents
1. Account Log in Log out and Time Out The user s account will remain active until 1 the user logs out of his her account by using the pull down box on the Main Menu to select either Default or another user or 2 the user closes the software A user account will also log out if the software System idle timeout is exceeded After 4 hours of inactivity the software account will automatically revert back to the Default user A screen will appear indicating that the time is about to expire with a 30 second countdown If the user elects CANCEL the clock with reset and the user account and application module will remain active for another 4 hours If the time expires the open application module will close returning to the Main Menu and the Default user Account Lockout User specific accounts can become locked out in several ways as noted below e Failure to change password within the allotted time e Incorrectly entering the password 99 consecutive times e The administrator locks a specific account Only the administrator level 10 can unlock a locked account This is done by using the Modify User entry in the Account Management module Note All accounts even the administrator can be locked if the incorrect password entry occurs as described above Change Password This module enables each user having an authorized account ID to change their respective password This option is grayed out2. Edt Configurabon Standard Curves Ip Liters Maral En 4 Show Prim Repon Show Stondord Curve Method Hoechsl 33258 dsDNA moore Show Peport Key Naigatinn Selecbon of thet Reuthon rl play Ehe current Report User s Manual version of this User s Manual is accessible from the Main Menu It can also be accessed by selecting from the Help pull down menu in any application module or from Start gt Programs gt NanoDrop gt ND 3300 V2 5 0 4 6 Section 5 Nucleic Acid 5 Nucleic Acid There are three NanoDrop coded applications within this module for determining nucleic acid sample concentrations e dsDNA 33258 Hoechst dye e dsDNA PicoGreen dye RiboGreen dye The user can also create and save other methods for analysis within this module by using the Method Editor see section 5 dsDNA 33258 Hoechst This application is configured to measure fluorescence of the Hoechst dye which is greatly increased upon binding to double stranded ds DNA Fluorescence of the Hoechst dsDNA complex is measured at 450 nm following excitation by the UV LED Be DE corpsen Curves Hau Foncurdung enini MTOE 17 54 PM Homsi 11258 delia Source U Miensurement Sample Load 1 io 2 ul oi your sample ened click the minsure bution 1 Sample mmm ID Sample P E
3. 30 00 195 20 00 i ss 000 RFU 495 87 0 00 80 500 520 540 560 580 600 620 640 660 680 700 007 Wavelength nm ng ml 50 000 Technical Service If after referring to the above troubleshooting tips you are unable to resolve your problem please contact your local distributor or NanoDrop Technologies for help The following information will be very helpful 1 Visual inspection of the light sources Check to see that the WHITE and or BLUE light sources are emitting when making a measurement Caution Do not look directly at the UV light source while making measurements To check the UV LED source hold a piece of White paper in front of the light source and look for a Blue hue on the White background 2 Application Module Screen Captures Screen captures of the actual spectrum as seen on your PC can be of great use in diagnosing problems Making a screen capture is quite easy Simply choose Save file from the pull down menu in the Header of the respective screen module This saves the respective image as a jpg in the respective module s folder in which the user is working The file can be sent as an email attachment to info nanodrop com 3 Data Archive Files If you have questions about your data please send the archive file containing the suspect data as an email attachment to your distributor or to info nanodrop com The archived file can be found at the following path C ND 3300 data gt U
4. 10 1 bc 10 1 11 Appendices asse eaiio 11 1 Instrument Specifications siseses a a a 11 1 Sample Retention System Solvent Compatibility 11 1 Section 1 Overview 1 Overview Instrument Description The NanoDrop ND 3300 Fluorospectrometer uses patented sample retention technology Surface tension The uniquely clean optics of the retention system combined with proprietary signal processing for the White LED applications enables measurements across a wide range of wavelengths using sample volumes of 1 2 ul without cuvettes and costly filter changes The excitation source comes from one of three solid state light emitting diodes LED s which are oriented 90 to the detector A 2048 element CCD array detector covering 400 750 nm is connected by an optical fiber to the optical measurement surface The spectrometer is configured with a cut filter to eliminate light transmission below 395 nm The intensity plots for each of the LEDs are shown below UV LED Blue LED White LED max 365nm max 470nm range 500 650nm equipped with cut filter equipped with cut uses virtual filtering that eliminates excitation filter that eliminates above 400 nm excitation above 495 nm 25 z Ir 20m A 1 Ta 25 C F 20m s 5 T Imer rar ES Relative Emission Intensity au Em pon Relativ
5. 08 450 nm Using 2 ul samples sensitivity for the Hoechst is approximately 150 picograms and the linear range is from 75ng ml to 1500 ng ml Details of a recommended protocol as well as performance data can be found at www nanodrop com A typical standard curve for this dye is represented in the figure that follows 3j User Default ly Are 12 55 dsDNA PicoGreen dye This application is configured to measure fluorescence of PicoGreen dye which is greatly increased upon binding to double stranded dsDNA Fluorescence of the PicoGreen dsDNA complex is measured at 525nm using the Blue LED excitation 9 1 Section 5 Nucleic Acid 455 Mensuromant Type 1 Sample In Minimum Gaia f 500 A AU 534 61 5 534 nm nalmi Using 2 ul samples sensitivity for the PicoGreen assay is approximately 2 picograms and the linear range is from 1ng ml to 1000 ng ml Details of a recommended protocol as well as performance data can be found at www nanodrop com A typical standard curve for this dye is represented in the figure that follows eget nut hud rs User Default 1 D 4 4 au 1 Th us 1 jam 1172 Maz B miyi 1155 ma lum ama 1280 1206 ame 10 aF 155 7 ara jim mas 60 9102 T 1442 14523 1843 fp jg 170 HESS maT ames xm 18 253
6. Basic Use The main steps for making a measurement are listed below A 4 y 1 With the sampling arm open pipette the sample onto the lower measurement pedestal 2 Close the sampling arm and initiate a measurement using the operating software on the PC The sample column is automatically drawn between the upper bushing and the lower measurement pedestal and the measurement is made 3 When the measurement is complete open the sampling arm and wipe the sample from both the upper bushing and the lower pedestal using a soft laboratory wipe Cleaning the Sample Retention System Wiping the sample from the upper bushing and lower pedestal as shown above upon completion of each sample measurement is usually sufficient to prevent sample carryover and avoid residue buildup Although generally not necessary 2 ul water aliquots can be used to clean the measurement surfaces after particularly high concentration samples to ensure no residual sample is retained on either the pedestal or the bushing It is recommended that the areas around the upper bushing and lower pedestal be cleaned i e with water after measuring a large number of samples This will prevent the wiping after each measurement from carrying previous samples onto the measurement pedestals and affecting low level measurements A final cleaning of all surfaces with de ionized water is also recommended after the user s last measurement Decontamination of Measurem
7. for the default user Note The administrator using the Options entry in the Account Management module establishes whether user passwords will expire and if so after how many days Passwords log file This file contains the User ID amp password for all accounts and is readable only by the software It can be found in the 3300 Datallog files folder It is strongly recommended that the administrator make a copy of that file and store it in the same log files folder as described above each time a new user account is added a password is changed If the administrator s account becomes locked the up to date copy can be renamed and used as the password log file 3 6 Section 4 Common Module Functions 4 Common Module Functions Module Startup Each module or application is accessible starting from the Main Menu All sample data will automatically be logged in the appropriate archive file Escape Key ESC The escape key 15 set to exit out of all screens Hitting the escape key twice will log the user out of an application module Basic Module Window Recording View User Default 5 Das 1 9 2006 2 02 PM Method PicoGmen dsDNA Source Blue Measurement Type Sample Make new BLANK Measurement Sample 5 100 0 Samples Units Y ng ml Reset Window RFU 525 nm 580 600 620 Wavelength nm Measure F1 Each time a software module is opened the Measure button is in
8. nanodrop It is strongly recommended that the password be changed after the initial account set up Any user can be set to a level 10 access although this is not recommended see Level 5 below Note The administrator or the last level 10 user account may not be deleted 3 5 Section 3 General Operation Level 5 is the security setting recommended for an ordinary user account account with this access will be password protected and will be able to select specific user preferences All data generated will be automatically archived in to the user s account in c ND 3300 data and the user specified location if that preference is selected Default level O security is the access level reserved for the Default account only This account enables any user without an account to access all active software measurement modules lt is not password protected however user preferences can be set for this account All data generated will be automatically archived into the Default folder within the c nanodrop data folder Note The administrator may disable the default user account for laboratories requiring unique user accounts User Access Manager _ x Modify User All Users Delete User Options Exit Full Name Level Active Locked Expired Expires Default user Active Not Locked Not Expired e Administrator 10 Active Not Locked Not Expired Never Mot Locked Not Expired
9. Cy5 Alexa647 All data is written to the archive file immediately upon completion of the measurement Inadvertent software or PC shutdowns should not affect the archive file Data Viewer Data Viewer is a versatile data reporting software program incorporated into the NanoDrop software that offers the user the ability to customize report structures import stored data and re plot data generated from NanoDrop instruments Using the Data Viewer is the most expedient method to review data This feature may be accessed during measurement sessions from the Show Report function found within each method module Accessing via the Main Menu is used to import and review previously archived data and also allows the software to be utilized as a stand alone feature i e when an ND 3300 fluorospectrometer is not connected to the PC 8 1 Section 8 Archived Data and Data Viewer 2300 2 5 0 User Default Li Users Manual Method User Editor Preferences Data Account Managemen Chan Password Data Viewer Features The Data Viewer is composed of two or three pages in a tabular form consisting of Plots Reports and Standard Curves where utilized The user may access any page by clicking on the tabs The software opens to the Plots page see Plots section below whether accessed through the Main Menu or Show Report Note Recording rather than Start Report must be selected in order to access the Data Vie
10. 0 10 2005 215 PM Measure 0 10 2005 215 PM Measure 3 4 Blank Default 5 Blank ES e D cic e gt a 2 gt gt gt Data Storage Hierarchy The hierarchy for archive files is as follows C ND 3300 Data gt User name gt Application Module Hoechst dsDNA PicoGreen dsDNA RNA RiboGreen etc User Defined Archive File Location In addition to the primary data storage users may elect to save their data to an additional location This option can be chosen under the Archiving Tab in User Preferences on the Main Menu by setting the Duplicate data storage box to On and then choosing the file path by clicking on the file folder icon under Duplicate Data Folder Save the alternative path by clicking on the Save Preferences button before exiting the User Preferences window 2 C ND 3300 4 Umbelliferone File Edit View Favorites Tools Help Back 2 y Search Key Folders EI Address C MD 3300 DatatDefaulti4 Methyl Umbelliferone v Go Folders 5 Date Modified C NanoDrop Data A 5 4 Methyl Umbeliferone 2005 12 05 v2 5 nfd 10KB NFDFile 12 5 2005 11 37 AM ND 3300 Data Sl 4 Methyl Umbelliferone 2006 01 09 v2 5 nfd 5KB NFDFile 1 9 2006 3 55 PM B Custom report Formats 3 O Default B 4 Methyl Umbelliferone 5 mu 555 Cy3 Alexa555 O 5 Alexa647 C
11. 8 Occurred at Open File This error message occurs when the user attempts to take a measurement while the data file is open Close the data file and you should be able to continue taking samples normally Insufficient Memory This error message or others with similar wording occurs when attempting to install the operating software on a computer that does not have at least 40MB of free hard disk space No Printer Connected This error appears when attempting to print when a printer is not attached to the PC It is non fatal and will not cause the software to shut down Sampling Concerns Sample Homogeneity The user must be sure the solution is homogeneous when measuring fluorescence Micro sampling from non homogeneous solutions can cause significant deviations in the fluorescence data generated Sample Accuracy and Reproducibility If you are obtaining results that seem inaccurate or not reproducible it could be the result of sample or aliquot non homogeneity or liquid column breakage It may be helpful to try the following to ensure representative results Clean the surface Make sure the sample surfaces are clean before starting the software module A dirty sample pedestal on startup can cause erroneous fluorescence measurements even negative values and signal saturation It is always a good practice to clean the sample surfaces with de ionized water to remove any dried sample that might be present 9 4 Use a 1 2 ul sampl
12. and then reconnected before selecting Retry You can confirm that the power management settings are correct by opening the Power Options Properties page by choosing Start gt Control Panel gt Power Options Both System Standby and System Hibernate should be set to never for the Plugged In column as shown below Power Options Properties 2 x Power Schemes Alarms Power Meter Advanced Hibernate this computer Note that changing the settings below will modify ud Select the power scheme with the most appropriate settings for the selected scheme m Power schemes Save Delete m Settings for Home Office Desk power scheme When computer is Plugged in E ue on Turn off monitor After 20 mins y After 15 mins y Turn off hard disks Never y After 10 mins y System standby Never y Atte 30 mins y System hibernates Never y After 45 mins y Cancel Static Electricity Discharge Discharge from the user to the instrument can be a problem in very dry environments It may be necessary for the user to wear a grounding strap to prevent the discharges from occurring Defective USB Port on PC If your instrument operates properly most of the time but the Connection Error appears intermittently it could be caused by the USB port on the PC If this occurs install the software and operate on another PC If the error does not occur on the second it may be nece
13. define the method type as nucleic acid protein or other Additional parameters set using this tab are e Graph minimum nm minimum wavelength nm for the x axis of the fluorescence spectral plot e Graph maximum nm maximum wavelength nm for the x axis of the fluorescence spectral plot 3 3 Section 3 General Operation Fluorescence tab allows for the selection of the LED source and gain settings UV LED excitation center point 365 nm Blue LED excitation center point 470 nm White LED 500 650 nm excitation AutoGain controls the length of excitation time based on fluorescent signal intensity Note The alternative to AutoGain 15 Fixed gain which sets the excitation time using a factor between 0 and 10 See section 4 for further details Edit method parameters Method name Edit method parameters Method name General Fluorescence Analysis Standard Curve Type 7 Polynomial order 1 Default Units J ug ml Analysis type J 1 pt General Fluorescence Analysis Standard Curve Type Jj Interpolation Default Units 9 ug ml Analysis type Formula 22 Fluor F a3 a4 F 34 Analysis nm 525 Al al A2 9 NaN ad Analysis tab gives the user the choice of determining concentration by reading off of a standard curve at one wavelength 1 pt analysis or by defining a specialized formula e Stan
14. following window Save Report As Choose how to save the report Choose this option to be able to reload the Full Report report into the Data Viewer at a later date Choose this option to export atab deliminited Export Report textfile ofthe displayed Report Table Table Only suitable for import into Excel Choose this option to export a tab deliminited Export Report amp textfile of the displayed Report amp Standards Standards Tables Tables suitable for import into Excel Cancel Using the Full Report option will allow the user to use the Data Viewer to reload the report at a later date The saved report may be recalled using the pull down Load Report If using the Load Report feature the report will be displayed with the default column configuration Note Access the User Preferences module on the main menu to modify and save preferred default configurations Reports are saved in an nfr format The other two options are meant for reports that are expected to be opened in Excel type spreadsheets To open these reports go to the C ND 3300 Data Reports folder and right click on the file of interest Additional features of the Report page Method Automatically populated with data method type Date and time Automatically populated when report is generated Report Name User defined designation for the current report Buffer mode Drop down box defining options for managing reports Max Buffer size Default number is
15. for a given calendar day are stored in a single archive file These files bear the name of the respective application module with the date appended For example an archived file entitled PicoGreen dsDNA 2005 10 10 nfd corresponds to PicoGreen assay data from the software session that was generated on October 10 2005 A unique file extension nfd has been given to these files to enable automatic startup with spreadsheet formats or by importing into Data Viewer see section on Data Viewer below The data may be edited and or reformatted and stored under names of the user s choice The spectrum can be re plotted from the wavelength data if needed for further analysis Note1 Fluorescence data shown in archive files are represented as they are displayed on the screen Note 2 For data from all modules a column entitled Measurement Type is included For each measurement this column will contain Measure Blank or Reblank If the value is Measure then the values in that row are from a normal measurement that has utilized the stored blank value If the value is Blank it indicates that the measurement is the initial blank recorded If the value is Reblank it is the re analysis of the previous measurement with a new blank PicoGreen d 2 Software 250 Sample ID UserID Date Time Measure T Gain Conc Units Min Gain RFL nm RELY al Delta 0 0 n n 0 10 2005 2 13 PM Blank 0 10 2005 214 PM Blank
16. set at the maximum of 200 o When the selected buffer size is reached the Report will either be Saved Cleared or Printed and Cleared o Selecting Show Report will bring the user to the report page All data is saved in the archive files and can be retrieved using the Import feature of the Data Viewer module Standards Page The Standards page will display the actual reference standards applied to each particular sample at the time of measurement Section 8 Archived Data and Data Viewer fie aporta Help Pots Report Standards Method Standards Opening Archived Data with spreadsheet programs The files are in tab delimited format and can be opened in Microsoft Excel or an equivalent spreadsheet program 8 5 9 Troubleshooting Error USB2000 Error USB2000 El Unable To find Device with Serial This error might appear upon software startup and usually indicates that the USB cable 15 not properly connected or the software 15 not loaded properly To troubleshoot do the following 1 Confirm that the USB cable is connected to both the PC and the instrument If the cable is connected properly but the error persists run the USB Reset application located in Start gt Programs gt NanoDrop gt Utilities gt USB Reset If USB Reset is not installed on your PC you can download it from the Downloads section of www nanodrop com 3 Follow the onscreen instructions fr
17. software automatically scale a plot 3 An additional option available by this dropdown is the choice of displaying either just the RFU plot or a spectrum showing the blank raw and RFU plots Note The spectrum with all data displayed uses two scales The right hand scale displays counts and is used for both the blank and raw data The left hand scale reports the difference between the blank and raw and is reported as RFU The two scales can be modified independently by overtyping the top value 4 The final option controlled by the Configuration dropdown is whether or not to display all available parameters on the main screen Default screen Exit This command closes all application modules and supporting options After clicking the Exit button the user has 10 seconds to cancel the exit command If no action is taken within 10 seconds the exit command is carried out Note All measurement data 15 automatically saved to an archive file and requires no user action 4 5 Section 4 Common Module Functions Show Context Help Context Help is enabled in the Main Menu all function modules and all Application modules The help feature is enabled by choosing Show Context Help from the Help menu or by selecting Ctrl H Once enabled placing the cursor on elements of the screen will automatically generate an explanation of that element Context Help remains active until the user deselects it Hoechst 33258
18. user may simply move the cursor over the plot of interest and click or use the Selected Plot drop down box which will also display the legend The selected sample will show up as a bold plot line Plots Sets Users may select the maximum number of individual plots up to 20 graphed per page Since a report can hold data for 200 samples and a graph page is limited to 20 plots additional sample spectra are displayed on new pages Each page is then referred to as a Set Legend Positioning the cursor over the legend box will bring up a visual display matching the sample name to a plot color The user is not able to select or highlight a sample from the legend Sample information Automatically populates with data associated with selected sample Data displayed is appropriate for data type chosen Note Information is based on data collected at the time the sample was measured and 15 not modified by a change in cursor position on the Data Viewer real time display Movable x and y axis available for all data types If the cursor is out of view in either direction rescale the axis by typing over one of the outer limit numbers The cursor RFU information displayed at the bottom of the page is determined by the position of the movable cursors The movable X determines the baseline from which the peak of the Y position is calculated Reset Baseline will reposition the x axis back to zero Reports The Reports page displays the data for select
19. you encounter a message stating that Windows has disabled remote access to this computer it will be necessary to run the Network Setup Wizard in order to set the permission levels on the folder Note 2 This error can also occur if the log files are set to read only To confirm right click each log file and select properties Deselect the read only box for each log file if it is checked Installer Error Incorrect command line parameters This error may be seen during the initial installation of the software and will be followed by the message Unable to locate the LabView Runtime Engine when the NanoDrop software is first opened To install the missing file go to C Program Files NanoDrop Utilities Run Time Installers and double click on the file LVRUNTIMEENG msi Follow all prompts to install at default location Other Software Error Messages An Error Occurred Code 7 Source Open file in This error occurs when any of the three log files dye list log passwords log user preferences log has been removed from the folder where the program files were installed C NanoDrop Data Log Files These files must be present in this folder in order for the NanoDrop software to operate 7 11 7 11 9 3 Can t find file OOIDRV INI This error occurs when trying to install the software without administrator privileges Contact your system administrator to install the software Error 7 O
20. 0 fluorospectrometer s measurement pedestals is easily addressed Simple wiping of the upper bushing and lower measurement pedestal with a dry laboratory wipe is highly effective This is possible since the measurement pedestal is in actuality the highly polished end of a fiber optic cable There are no cracks or crevices for residual sample to get trapped within Sample Homogeneity Sampling from non homogeneous solutions particularly when using small volumes can cause significant deviations in the data generated using all measurement technologies including fluorospectrometry Genomic DNA lambda DNA and viscous solutions of other highly concentrated nucleic acids such as re suspended nucleic acid preparations are common examples known to the molecular biologist Proteins are subject to denaturation precipitation and aggregation and therefore may require special handling to ensure sample homogeneity Bubbles must also be avoided since they can lead to erroneous signals When dispensing samples it is advisable not to dispel the full liquid sample content from the pipette tip and risk bubble introduction to the sample being measured Effect of Evaporation and Solvents Evaporation of the sample during the measurement cycle usually has just a minimal effect on readings for aqueous samples Highly volatile solvents such as hexane will likely evaporate before the measurement can be completed Less volatile solvents such as DMSO can be used successf
21. 2 NanoDrop ND 3300 Fluorospectrometer V2 5 User s Manual NanoDrop Technologies Inc 3411 Silverside Road Bancroft Building Wilmington DE 19810 USA Voice 302 479 7707 Fax 302 792 7155 Email info nanodrop com www nanodrop com NanoDrop 15 a registered trademark of NanoDrop Technologies Inc Other parties trademarks are the property of their respective owners and should be treated as such Copyright O 2006 NanoDrop Technologies Inc rev 3 06 TABLE OF CONTENTS AO AA a M 1 1 Instrument Description ocooonnnccconnncccnconocononconononcnnaronononcnnonannnconananenonens 1 1 Virtual Filtering White LED cccococcccoconoccccononocanonnononononnnononanonnnnos 1 1 A T 1 1 ADPIC ORNS uk ut id eli Lu tue ted Eee 1 2 wir E 1 2 2 Initlal SG UD oet ie on e ced oe o dictionem educi ce 2 1 Computer REGQUIFEMEMIS iii 2 1 Software Instala A rovs tp C Eo eut at 2 1 Software Upgrades i pice Deed eoo leen eda ues cose ibas a eds esu DPA ILES uds 2 1 Registering Your Instrument ss iio iier Eco e RE 2 2 3 General Operation siii iia 3 1 The Sample Retention 3 1 Cleaning the Sample Retention 3 1 Software Architecture and 3 2 Method Rees a 3 3 BULUM ce
22. 5 Ot 19911 Gane D cedar A ee RNA RiboGreen dye This application is configured to measure fluorescence of RiboGreen dye which is greatly increased upon binding to RNA Fluorescence of the RiboGreen RNA complex is measured at 525nm using the Blue LED excitation A A eee oe _ FINA Source Mensuroment Type Exondard 3 Binko new LANE Mirnsurmement AU Sy 4587 8 533 nm 459 100 0 Using 1 5 ul samples sensitivity for the RiboGreen assay is approximately 7 5 picograms It is best to use two distinct dye stock working concentrations to cover a dynamic range of 5 ng ml to 1000 ng ml Details of the recommended protocol as well as performance data can be found at www nanodrop com A typical standard curve for this dye is represented in the figure that follows 5 2 Section 5 Nucleic Acid Fisimence OW Sdad o 53 Sdad 5000 1203 Sdad 12500 4 2000 Section 6 Protein 6 Protein The single NanoDrop coded application within this module for determining protein sample concentration uses the Quant iT system from Molecular Probes This protein dye is a fluorescent stain used to quantitate minute amounts of protein in solution The dye is excited using the Blue LED and has an emission wavelength range of 590 610 nm Miengurement Sample Sample QunaT4T P
23. a Preferences Exil L upiscabes Diets I dor Duplicate Data Storage Primer date storage r hayi enabled end lobaled al CADE3360 Dain Custom Repara Mnihind non 1 50 TAL 1 defeat report darme nf PieeGreen HOMA Pi MC 3300 defecit ropat sonat n Toggle PubeGraen PHA r4 HD 3300 default repart format Quant Protein TR defecit repart torment nfi Report Moth Limbelderano Ri HD 3300 defeat nf yh A reu HD 3300 delia repart farei Change Cy Almah TR HO T30 detect report torment nf Custom Pluanseooin FITC TA HE 2300 dale opor Somos nt Report sullati TRHU MO 3300 defert repart Duplicate data storage The duplicate data storage option is selected using the User Preferences window Account Management The Account Management module provides options for directing where specific data files are archived allowing users to segregate their data into personal folders The Account Management module is accessible to the administrator and level 10 users only The module will be out for all other users Account Types There are three types of user accounts Level 10 is the highest security setting and any level 10 can add new users modify a user delete a user and set password options At the time of software installation the only level 10 account is Administrator whose initial password is
24. active as noted by its grayed out appearance blank must first be measured before the Measure button will become active Note For all methods that use the White LED excitation source Initialize replaces the Blank Reblank function The Measure button is used to initiate the measurement sequence for all samples non blanks lt is activated by depressing the F1 key or clicking the Measure button Blank F3 Before making a fluorescent measurement a blank using the buffer solvent or carrier liquid the sample of interest is in must be measured and stored It is not necessary to blank between every sample Re blank F2 The Re blanking option F2 establishes a new reference blank that is used for the calculations of subsequent samples However unlike the Blank F3 function the Re blank feature recalculates the fluorescence spectrum for the most recent sample and displays this on the screen When the Re blank function is used the following message appears Blank Applied To displayed Spectrum Initialize White LED When working with applications that use the White LED the Blank and Re blank buttons will be replaced with a single one called Initialize Before making a fluorescent measurement utilizing the White LED source the module must first be initialized and the source s emission stored The stored information is required for the Virtual Filtering algorithm Measure CO Recording Initialize Method C
25. ain Networking Windows 2000 Professional XP Home and XP Professional using using a server Workgroup Networking On the Security tab set he permissions for On the Sharing tab select share this folder as the Sharing tab set the Network sharing Users to Full Control as shown below shown below and security settings as shown below 4 0 1 Fropertios HansDrop 3 0 1 Properties ax Con faeit Diria GM eh cdo pes ca iiri ol Fei coss TOR Chin fou can share the folder aom olher ures on aur gus rie v Errei earen ade Pera Lens oet nem To ks Uri fodder cheb Share Eo ha thng hari a mae obs ard io subis Xl is peu bares mieti ri check bane l Dip rok Parta Holds i ahasa that cunt She rime CEEL To dues Ha o with Lob ii ore a ite col Eas bret check E loner haee ae Mala 201 5h 3 ET Lti Ai rik uid t Lo harg Ta ret permirtionz for hoe uraa Bi AAA falda ovem rara Porron Don e hase Chen holes ons Ehe nescis Witte Ta cone Caching For a advanced thes shared okdes chek Cachan Les chal Armed o j x cm Note 1 If
26. ccurred at New File This occurs when the C ND 3300 Data file folder has been removed from C drive or it has been corrupted this message appears click on the Stop button in the error wndow Close the NanoDrop Fluorospectrometer software Open Windows Explorer and create a new folder on the drive Name the folder C ND 3300 Data case sensitive and close Windows Explorer Click on the arrow in the far upper left of the NanoDrop Fluorospectrometer software Restart the NanoDrop Fluorospectrometer software Can t Find LabView RunTime Engine This error message likely means that one or more of the software components have been removed or corrupted If this occurs reinstall the NanoDrop software using the installation CD or download from www nanodrop com EZUSB SYS Cannot Found this error message appears do the following Windows 2000 type C WINNT INE in the file path text box Windows XP type C WINDOWSI INF in the file path text box This should allow the software to complete the installation successfully Driver X Configuration Failed You Must Manually Edit the Registry This error message or others with similar wording appears when attempting to install the operating software on a computer running Windows 2000 or XP It occurs because the user does not have the necessary authorization to install the software on the computer Contact your system administrator if this message appears Error
27. centration of the sample of interest Note 1 The module will always output RFU relative fluorescent units even when a standard curve is not generated Note 2 Be sure to select the preferred concentration units before running the first standard Changing units after a measurement will result in the loss of the standard curve data Method Specifies the current application Units This term refers to the way a sample concentration is expressed based on a standard curve It is only populated when a standard curve is utilized The operator can choose which of six unit configurations to use in several ways The method editor allows a unit choice as a default selection while the method page allows for interactive changes in the selection The units can be changed once a sample has been run by selecting the Reset Window button Note Changing the units will clear all standards Itis important to select the units of interest when generating standard curves before measuring the first standard Actual Gain Auto Fixed Gain are the two options for setting the gain or excitation time When the Auto Gain default mode is active the source LED illumination excitation time is optimized for each sample The Actual Gain value for the respective sample will be displayed in this mode A value of 10 0 corresponds to the maximum gain or excitation time maximum of 1 second 1000 msecs 4 3 Section 4 Common Module Functions The Fixed Gain optio
28. d on your PC you can download it from the Downloads section of www nanodrop com 6 Click on the Device Connected as shown below If more than one USB device is connected view each of them One of the connected devices should give an idVendor and idProduct as 0x2457 and 0x1002 respectively as shown below If these are present the USB function of the instrument should be OK If the dVendor and idProduct are different than below or if no USB device is present in the list go to step 7 File Options Help My Computer Device Descriptor ntel r 82801DB DBM USB Universal Host Controller 24C2 bcdUSB 2 RootHub bDeviceClass bDeviceSubClass Port1 NoDeviceConnected bDeeicsPrutocdl Port2 NoDeviceConnected azPRacketSizel Intelfr 32801DB DBM USB Universal Host Controller 24C4 idVendor RootHub Port1 NoDeviceConnected Port2 NoDeviceConnected Intel r 32801DB DBM USB Universal Host Controller 2407 RootHub Port1 NoDeviceConnected Port2 NoDeviceConnected iManufacturer iProduct iSerialNumber bNumConfigurations ConnectionStatus DeviceConnected Current Config Value Intel r 32801DB DBM USB 2 0 Enhanced Host Controller 24CD Device Bus Speed RootHub Device Address Open Pipes Port1 NoDeviceConnected Port2 NoDeviceConnected Port3 NoDeviceConnected bEndpoint ddre 0x02 Port4 NoDeviceConnected Transfer Type Bulk Endpoint De
29. dard Curve Type determines the default standard curve fitting method Choices include o Interpolation linear interpolation between adjacent standards o Polynomial order 1 2 or 3 fits the standard measurements with 1 e Default units there are six choices of concentration units 2 or 3 order polynomials Note The operator may also change the standard curve fit type using the toggle button on the standard curve page within each module Source Correction tab an additional window available when the user selects the White LED using the Fluorescence tab Edit method parameters Method name Cy3 Alexab55 General Fluorescence Analysis Source Correction For White LED illumination the virtual emission filter interval AX is symmetrically applied around each Analysis nm emission wavelength selected All RFUs outside of the virtual filter interval are setto zero The Default interval is 20 nm settable in the table below Note it can also be modified within the respective fluorescence method module Emission Windows Analysiis nm Emission filter interval A 565 20 3 4 Section 3 General Operation Settings include e Analysis nm emission wavelength or wavelength formula of optimal fluorescence intensity e Emission Filter interval AX symmetrically applied around the analysis nm emission wavelength selected Note 1 The analysis wavelength cursor can be man
30. e Emisi n Intensity y gt PERS 300 350 400 450 00 250 600 350 400 450 500 530 600 630 Wavelength A nimi Wavelength Virtual Filtering White LED In order to excite a broader range of fluorophores 7500 650 nm range a White LED source is selected The White LED is composed of a Blue LED and a yellow orange phosphor excited by the Blue LED to give an approximately White spectrum typically bluish in appearance Virtual filtering patents pending involves taking a reference spectrum as a wavelength intensity map of the White LED yellow peak The reference spectrum is then normalized to a non fluorescence wavelength of the sample s signal spectrum This normalized reference spectrum when subtracted from the signal spectrum will remove nearly all the stray excitation contribution to the fluorescence signal spectrum The normalization or scaling is typically done at a wavelength within the stability envelope the region of the White spectrum on the short wavelength side of the sample s fluorescence peak where the fluorescence signal is less than 5 of its peak value This is illustrated in the figure below Initial Reference Spectrum Total Fluorescence TU Normalized Reference Spectrum Sample Fluorescence One of the factors making it feasible to extract the Sample fluorescence by subtracting a scaled representation of the source is the inherent off angle light rejection properties of the op
31. e size Very strange results can occur when the liquid sample column is not completely formed during a measurement While making a measurement visually confirm that the liquid column is formed If necessary try 1 5 2 ul samples to ensure the column is formed Also proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form If this occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 20 times This will re condition the surface allowing for the liquid sample column to form Unusual Spectra For particularly low fluorescence a non uniform spectral profile can result from a change in ambient light hitting the detector relative to the Reference originally obtained during blanking Mercury vapor lighting may be give rise to a 546nm peak and 610nm as seen in the first image below Often re blanking will correct the ambient light difference dsDNA 33258 Hoechst Xx File Edt Standard Curves Show Context Help gt Print Screen Recording 1224 Standard Cuve User Dota Method dsDNA 33258 Hoechst Source UY Measurement Type Standard 4 Using 2 ul load up to 5 replicates of your th standard and measure Autoscale Silent Replicate 4 100 00 30 00 eal td ng ml 50 000 10 0 Units ng ml 7000 B M 50 00 0 0 Measurements 40 00 00 a
32. ed samples in a table format The user may modify column configurations for each method type and save multiple customized formats by using the drop down Report options 8 3 Section 8 Archived Data and Data Viewer 1 configuration editor Select the report columns to display and the onder That the columns should appear Change Mom Prampban ud Man EE the order by selecting and dragging a column Uum Dum Go im hoader string to the desired position Report columna Move Up Move Down Report tool bar drop down options include Sort Allows users to sort data by column example by date or sample name Save report format Saves specific report in appropriate data type folder All files saved with nfd extensions even if all files is chosen To designate a saved report format as the default format exit to the Main Menu choose Users Preferences and click Change settings A list of the various saved formats available for the specific method type will be displayed Report Format Allows saved report formats to be loaded either before or after data is imported Print report Will print out only the Report page by default Users may choose whether or not to print out the standards or plots pages by selecting these options under the Configure drop down on the tool bar Save Report and Load Report There are several choices for this option as seen in the
33. ent Pedestals If decontamination is necessary a sanitizing solution such as a 5 25 solution of sodium hypochlorite bleach freshly prepared can be used to ensure that no biologically active material is present on the measurement pedestals The metal fiber optic fittings are made from 303 stainless steel and are resistant to most common laboratory solvents see the Solvent Compatibility appendix 3 1 Section 3 General Operation Special Cleaning Requirements for Proteins Proteins and solutions containing surfactants can un condition the measurement pedestal surfaces so that the liquid column does not form well with 1ul samples If this occurs buff the measurement pedestal and upper bushing by rubbing each with a dry laboratory wipe 25 50 times This will re condition the surfaces allowing the liquid sample column to form Sample Size Requirements Although sample size is not critical it is essential that a liquid column is formed and the gap between the lower measurement pedestal and the upper bushing is bridged with sample It is best to use a precision pipettor 0 2 ul with precision tips to assure that sufficient sample 1 2 ul is used Lower precision pipettors 0 10 ul and larger are not as good at delivering 1 ul volumes to the measurement pedestal If you are unsure about your sample characteristics or pipettor accuracy a 2 ul sample is recommended Sample Carryover Prevention of sample being retained on the ND 330
34. fluorophores that can be measured using the ND 3300 along with the most appropriate excitation LED 365nm 470nm UV LED Blue LED White LED 40 nm 10 soonm dis UV LED BLUE LED WHITE LED GFP wt Em508 GFP wt Em5 8 Alexa 555 Em565 Quinine Sulfate Em 450 EGFP 509 568 EmboO Hoechst 33258 Em450 FITC FAM Em515 4 EMBAT 4 MU Em450 Alexa 488 Em520 Sulforhodarnine 101 Em amp 0D Dots Various PicoGreen Em525 S CTMR Em570 RiboGreen Em535 Dots Various Alexa 555 Em565 TET Em535 Emission collected B Phvcoendhrin Eris 5 995 between 400 750 nm Dots Various Patents The sample retention technology used in the ND 1000 Spectrophotometer amp ND 3300 Fluorospectrometer is covered under US patents 6 628 382 and 6 809 826 Other patents are pending Section 2 Initial Set Up 2 Initial Set Up Computer Requirements The NanoDrop software will only run on an IBM compatible PC meeting the below criteria Mac versions of the software are not currently available e Microsoft Windows XP or 2000 operating system The operating software will not work with Windows 95 98 ME or Windows NT 233 MHZ or higher processor CD ROM drive 32 MB or more of RAM 40 MB of free hard disk space Open USB port the instrument can only be connected the USB port Microsoft Excel or other spreadsheet program to manipulate archived data optional Software Installa
35. ght emitting diodes LEDs e Excitation Maxima of LEDs UV 365 nm Blue 470 nm White 500 650 nm Light Source three light emitting diodes LEDs Detector 2048 element linear silicon CCD array Wavelength Range 400 750 nm Wavelength Accuracy 1 Wavelength Resolution 8 nm FWHM at Hg 546 m Fluorescence Precision lt 5 CV 10 nM fluorescein Measurement Cycle Time 10 15 seconds Dimensions 20 cm X 15 cm x 12 cm Weight 3 Kg Sample Pedestal Material of Construction 303 stainless steel and quartz fiber Operating Voltage 5 all power supplied by USB port Operating Power Consumption 2 W Standby Power Consumption 1 W CE and UL CSA Approval Included in system software compatible with Windows 2000 or XP Sample Retention System Solvent Compatibility The NanoDrop ND 3300 Fluorospectrometer is compatible with most solvents typically used in life science laboratories These include methanol ethanol n propanol isopropanol butanol acetone ether chloroform carbon tetrachloride DMSO DMF acetonitrile THF toluene hexane benzene sodium hydroxide sodium hypochlorite bleach dilute dilute dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable
36. hyl umbelliferone 4 MU This module is configured to measure the fluorescence of enzymatic cleavage products containing 4 methyl umbelliferone at 460 nM using the UV LED Detection of 4 methyl umbelliferone may be used as a sensitive quantitative assay for b galactosidase or other enzymes that cleave substrates linked to 4 MU Cleavage of 4 methyl umbelliferyl b D galactoside by b galactosidase yields the fluorescent molecule 4 methyl umbelliferone 7 hydroxy 4 methylcoumarin 4 MU Note 4 methyl umbelliferone is fluorescent only above pH 8 A representative spectrum of 4 MU diluted in 0 1 mM carbonate buffer is shown below 7 2 Section 7 Other Samples 4 Sauren LW Messuremunt Type 1 Garnple Lond to 7 ul od your sample and chick the measure bution Sample io 160 0 ILE Using 2 ul sensitivity has been shown to be 2 picograms or 1 pg ul Performance data can be found at www nanodrop com 7 3 Section 8 Archived Data and Data Viewer 8 Archived Data and Data Viewer Sample data from all application modules is automatically stored in archive files and can be opened by either the integrated Data Viewer software program or spreadsheet programs such as MS Excel Archive File Creation Every time an application module 15 started an application specific archive file 15 created for the user that is logged in measurements made by the user in that application module
37. ich the residual signal with scaled background compensation removal is displayed and calculated It is symmetrically applied around the Analysis nm emission wavelength selected All fluoresence readings outside the virtual filter interval are set to zero The Default interval is 20nm and may be modified using either the Method Editor module or from within the respective fluorescence method module See section 3 for further details Note The baseline for non analyte non fluorescent samples can exhibit significant non uniformity and irregularity with the virtual filtering algorithm The greatest effect is on the detection limits Sensitivity for low concentration fluorophores Fluorescence of concenirated solutions where the fluorescence intensity is significantly greater than the non analyte background fluorescence can be measured with good reproducibility Working with more concentrated solutions for fluorophores such as Cy3 or Alexa Fluor 555 where the excitation maximum 555 nm is also the maximum emission of the White LED can overcome the baseline non uniformity Configuration There are four options that are controlled by the Configuration tool bar pull down feature as shown below B dsDNA 1 The fluorospectrometer has a default setting of making a beep noise at the end of each measurement The user may choose to silence this operation using the Configuration feature 2 The dropdown allows for the option of having the
38. le 3 5 BESTIA Mandal TE ad 3 5 Srl ie Lites eU C EA UU ede 3 5 Account Management ccccssececceseecceeseecceuseeceeseecseseesseueeesseseesseaeees 3 5 iara tocan deeton aT 3 6 4 Common Module Functions 4 1 Module Stan id PL 4 1 EScdpe ey IES lor at oa ru 4 1 Basic Module WIN LOW iius evel ean eee eed 4 1 Measures al 4 1 A i mde idet 4 1 micas AS RE T S MH PS 4 1 Initialize White LED e eere E 4 1 Print Screen rn 4 2 E dto tol 4 2 nisle rss LE pinitos 4 2 Saving Current Screen as jpg 4 2 a Xdax 4 2 Prititardddgve MOLU iu sedi sadi as eina edu DM dues od 4 2 SNOW sisi 4 2 View Standard Curve cooooccccncccccconncccnncconononnccnnnnconnnannnnnnnononnnnnnnnnnnnnnnnnanenons 4 2 Measurement TD Auten 4 3 PRU p ese eck a sce ee 4 3 neice Matte acca edad Stace Mu LL MAE mE PEU 4 3 GAN PEDE NL 4 3 Minimum Maa 4 4 Sara o e a dl a 4 4 4 4 4 4 a toda TRE TORIS 4 4 yr PE RS 4 5 SORT UN RE DET 4 5 unu M
39. n is selected when constant gain or excitation time is wanted for every sample Fixed Gain settings range from 0 1 to 10 0 in 0 1 increments Note 1 RFU signals for all gain settings are normalized for the actual time of measurement Note 2 The Auto Gain feature 15 activated or deactivated via the Method Editor module Minimum Gain This feature will appear when the Auto Gain feature is selected This is a selectable option which alerts the user when the minimum amount of time a measurement should collect data as set in the method Editor has not been met Saturation nm The NanoDrop software automatically limits the amount of time the LED source is on in order to prevent too much light from saturating or flooding the spectrometer detector for all wavelengths above the user selectable setting termed saturation nm The default setting is 490 nm The user may find that for a particular application choosing a saturation nm nearer the emission nm of interest may result in longer excitation times and higher sample fluorescence readings The software will not prevent saturation for wavelengths below the selected saturation nm Note 1 The fluorescence signal from the sample fluorophore emission wavelength is never allowed to saturate the detector when the Auto Gain setting is active Note 2 When Fixed Gain is active i e when the user defines the excitation time it supersedes the Saturation nm setting This can result in saturation at any
40. naturally fluorescent After covalent attachment of FITC to the molecule of interest FITC labeled reagent must be separated from unbound fluorphore before taking a measurement A representative spectrum of a 0 5 uM 500 nM Fluorescein sample diluted NIST SRM 1932 measured on a NanoDrop Fluorospectrometer using the Blue LED follows lecnrding View Laar Dresinull 1 9 2006 2 46 Mensureman H Lond 1 to 2 sl ol your sample asd click Bus bation Sotureston mm 498 Dam 1 ns L RF 515 554 b 515 nm HA 4 nihi Linearity has been demonstrated between 0 1 nM to 500 nM Performance data can be found at www nanodrop com Cy3 Alexa Fluor 555 This module is configured to measure the fluorescence of Cy3 or Alexa Fluor 555 at 565nm Unlike the Nucleic Acid amp Protein dyes described earlier these fluorophores are naturally fluorescent After covalent attachment of the fluorophore to the molecule of interest the fluorophore labeled reagent must be separated from unbound fluorophore before taking a measurement 1 00 317 Ph Sample ih MO AL 565 1563001 20 3i 565 mm 1510 Using 2 ul sensitivity has been shown to be 5 femtomoles or 2 5 nM Linearity has been demonstrated between 2 5 nM to 3000 nm Performance data can be found at www nanodrop com 7 1 Section 7 Other Samples Cy5 Alexa Fluor 647 This module is configured to measure the fluo
41. ns respectively 4 2 Section 4 Common Module Functions File Edit Configuration zs E Help Save as Load Measure Re View Steaderd Curve Selecting the View Standard Curve button allows the user to view the standard curve at any time An example is shown below phe Shoe APL 2 Wud AMIS User Dehoull Reteence 000 10 3 06 E 15 1 1 4 20 Shandad 1 1172 32 m a 3 53 1133 2 2 5 2099 2250 23006 2090 esL rum Standmd I 2 4174 02 n am Slamkmd 4 uas Hun nas es Standaid 5 600 Standard Ha 022 E Mod Eos am amo Ta Note 1 The user should select the proper concentration units for the respective assay on the basic module window before generating a standard curve as changing the units after a measurement will result in the loss of the standard curve data Note 2 The user may exclude points by selecting the data point to be deleted in the table and then clicking on the Delete Point button Additional module functions Measurement Type The user must select the type measurement i e reference standard or sample before each aliquot is read If desired a standard curve can be created to calculate the concentration for sample unknowns Accordingly the user would select Reference for a non analyte solution Standard for a known concentration of reagent and Sample for an unknown con
42. om USB Reset After unplugging and then reconnecting the USB cable the Found New Hardware Wizard should start as shown below Windows XP SP2 operating system will ask to allow it to search the internet for the proper software as shown Select not this time Follow the prompts for automatic installation of the software Found Mew Hardware Wizard Found Mew Hardware Wizard Welcome to the Found New Hardware Wizard vell search los current and upiared role by locking on pour computer on she Fardeane inttallation CD on Ba Wree Ugia La ads eth yon parikan Bead qur pa hii wean vena rl str lor ManeDuop USI Device H pour hadame come with an installation Cf ue Peppy disk M nose Wali fara frd ba batch sola es bene ony C Tet noe and gray ire connect atesi do you want ihe bo cot E not than tma rect thes sota om i cornu li Front o Inala hora heal af beta oco abcr Hasi bo continue JL nes cm Intro Page Windows XP SP2 Other Windows Operating Systems 4 Try the NanoDrop software if it works properly you are finished If it does not operate properly go to step 5 5 Shutdown the NanoDrop software and open the USBView utility to confirm proper USB communication Start gt Programs gt NanoDrop gt Utilities gt USBView If USBView is not installe
43. om the File pull down menu accessible within all modules Eg Edit Configuration Standard Curves Help Page Setup Print Window CU ink Print Screen 8 Save Window k Print Report A Curve Exit Ctrl Q The Print Window option and Ctrl P enable selection of printers available to the respective user By selecting the Save Window option the user may save the current window as a jpg image If the PC is not connected to a printer you will see an error message that will not cause the software to shut down Show Report F7 The user can display the entries comprising the current Sample Report at any time by selecting the Show Report button This function will enable the Data Viewer software described in section 10 Parameters specific for the individual application modules are populated for each individual Sample ID View Standard Curve All applications or modules include an optional Standard Curve functionality In addition to the measured RFU output the user can generate a standard curve using between 1 to 7 standard concentrations 1 to 5 replicates per point Note When a valid standard curve has been established the solid green button will be replaced as seen below File Edit Configuration Standard Curves Help File Edit Configuration Standard Curves Help A standard curve can be saved and reloaded using the Standard Curve pull down menu and choosing the Save as or Load functio
44. oubleshooting section for possible solutions Configuring the System Font The NanoDrop software is designed to look best with the MS Sans Serif font 8 point To check that the system font is set to the proper selection 1 Open the Displays Properties by right clicking on the desktop and select Properties gt Appearance Additional step for Windows XP click on the Advanced button 2 From item list select icon 3 Select the MS Sans Serif western font and select 8 point 4 Click OK Other selections can be used but may either cause some text in the NanoDrop software window to not fit well or result in the function selection tabs across the top to become inaccessible Software Upgrades NanoDrop Technologies makes periodic upgrades to the NanoDrop software These upgrades are available for download at www nanodrop com 2 1 Section 2 Initial Set Up Cable Connection To make measurements with the instrument simply connect the USB cable to the back of both the instrument and the PC Registering Your Instrument Please register your product We periodically update our software and add new features free of charge We would like to keep our user list updated so that we may alert you to these upgrades All information supplied to NanoDrop Technologies is completely confidential You can register at www nanodrop com 2 2 Section 3 General Operation 3 General Operation The Sample Retention System
45. ples This will bring up a new window with an Import Folder box and a Directory Tree Selected Samples D De Features include Import folder Used to select folder where data is to be imported from Folder selection must be at the level of user or higher Directory tree Used to select specific data to be imported Clicking on the square to the left of each file name will provide further detail to each level Users may choose to select either individual samples within a file or the entire file All import selections must be of the same application or method type Section 8 Archived Data and Data Viewer gt gt gt or lt lt lt Used to move the highlighted sample choices to or from the Selected Samples box Note The software defaults to a buffer size of 200 samples Search Function allows the user to locate specific data by searching through sample ID names Note This function is case sensitive Sample Information and Spectrum Are populated with the information associated with the most recently highlighted sample Import and Return Uses selected sample data to populate Plots and Reports windows and then returns to the Plots window Plots The Plots page is where selected sample spectra are displayed Features include Method Auto fills in module name Date Auto fills in date and time of Selected Plot There are two methods of selecting or highlighting individual sample data The
46. rescence of Cy5 or Alexa Fluor 647 at 670 nm Unlike the Nucleic Acid 8 Protein dyes described earlier these fluorophores are naturally fluorescent After covalent attachment of the fluorophore to the molecule of interest the fluorophore labeled reagent must be separated from unbound fluorophore before taking a measurement An example of Alexa 647 is displayed below Cyri de Caefuaranon asia fue Help 1 5 2986 111 Pu ew Mothod Alenia EDT NE Marmsuremmmt Sample Land T to Z ul id your and chick the measure bunon Sample iD AX H T Using 2 ul sensitivity has been shown to be 5 femtomoles or 2 5 nM Linearity has been demonstrated between 2 5 nM to 3000 nm Performance data can be found at www nanodrop com Quinine Sulfate This application is configured to measure the inherent fluorescence of Quinine Sulfate Fluorescence of Quinine Sulfate is measured at 450 nm following excitation by the UV LED Messen Purhlssk Prim tenen Reca Leer B Print Repo Standard Cure 62172005 4 48 uininn sulin s Type Sampler Lond 7 wl al your sample nad click the momeure bution eram am so se ue Using 2 ul sensitivity has been shown to be 60 picograms of quinine sulfate Linearity has been demonstrated between 30 ng ul to 3000 ng ul Performance data can be found at www nanodrop com 4 Met
47. rotein ID 89 600 nm 11619 Using 2 ul samples sensitivity for the Quant T assay 15 approximately 0 05 micrograms It is best to use two distinct dye stock working concentrations to cover a dynamic range of 0 05 micrograms to 5 micrograms BSA Details of the recommended protocol as well as performance data can be found at www nanodrop com A typical standard curve using the recommended high concentration working stock for this dye is represented in the figure below RFU Aui 2 Wua a AS User Default en E T 7 j E 1 17107 A 55 as Gir EE 3 Uu 1065 nea MOT 27533 2993908 E A7 aa METE CEA EN a 556307 SA7 55d 5242079 QUOD 6505 622504 ERE 5000 000 x06 000 ua 6 1 Section 7 Other Samples 7 Other Samples There are five pre coded applications within this module Fluorescein FITC FAM Cy3 Alexa555 Cy5 Alexa647 Quinine Sulfate 4 methyl umbelliferone The Cy3 amp Cy5 fluorophores and their respective counterpart methods utilize the White LED excitation source Both quinine sulfate and 4 MU methyl umbelliferone are excited by the UV LED while FITC fluorescein uses the Blue LED source Fluorescein FITC FAM This module is configured to measure the fluorescence of Fluorescein FITC at 515nm Unlike the Nucleic Acid amp Protein dyes described earlier Fluorescein is
48. scriptor wMaxPacketSize 0 0040 Port5 NoDeviceConnected Es 0x00 Port6 DeviceConnected Generic USB Hub Port1 NoDeviceConnected Endpoint Descriptor Port2 DeviceConnected NanoDrop USB Device bEndpoint ddress 0x00 Port3 NoDeviceConnected Transfer Type Control Pod NoDeviceConnected wMaxPacketSize 0x0507 1287 Devices Connected 2 7 Install the instrument on another PC to rule out a faulty USB hub port on the original PC Run USBVIEW on the 2 PC If the same behavior is exhibited then the instrument may need service Contact NanoDrop Technologies or your distributor if this occurs 9 1 Connection Errors Connection Error There was an error communicating with the instrument Try the following Check the USB cable and select Retry If Retry fails disconnect the USB cable and then reconnect and select Retry again If this does not solve the problem refer to the Troubleshooting section of the User s Manual available from Main Menu This error occurs whenever the USB connection is disrupted while operating a software module In most cases selecting Retry will reconnect properly Some possible causes and solutions are listed below Power management scheme on the PC The USB communication will be lost whenever your PC automatically goes into standby or hibernate mode Retry will NOT reconnect the instrument If this occurs the USB cable will need to be disconnected
49. sername gt Application Module 9 5 Section 10 Maintenance and Warranty 10 Maintenance and Warranty Cleaning The primary maintenance requirement of the NanoDrop ND 3300 Fluorospectrometer is keeping the measurement and optical surfaces clean Upon completion of each sample measurement wipe the sample from the upper bushing and lower pedestal to prevent sample carryover and avoid residue buildup It is also recommended that both measurement surfaces be cleaned with de ionized water upon completion of a testing series No other regular maintenance is required Decontamination of Measurement amp Optical Surfaces If decontamination is necessary a sanitizing solution such as a 5 25 solution of sodium hypochlorite bleach freshly prepared can be used to ensure that no biologically active material is present on the measurement pedestals The metal fiber optic fittings are made from 303 stainless steel and are resistant to most common laboratory solvents see Solvent Compatibility appendix Warranty All fluorospectrometers and accessories manufactured by NanoDrop Technologies are warranted against manufacturing defects in parts and labor for a period of one year Additional one two and three year warranty extensions are available Plan descriptions can be found at the following link www nanodrop com Section 11 Appendices 11 Appendices Instrument Specifications e Sample Size 1 2 microliters e Light Sources li
50. ssary to replace the USB card on the original PC If none of the troubleshooting steps above solve the problem refer to the Technical Service section for getting help from your distributor or NanoDrop Technologies Error Code 8 Error Code 8 Error reading a configuration file This is most likely caused when a user does not have write access to the folder containing the NanoDrop operating software usually found in CAprogram files Contact your PC administrator to share this folder with Read and Write permissions for all users For more details referto the Troubleshooting section of the User s Manual accessible below OPEN USERS MANUAL BEFORE EXIT EXIT 7 11 This error occurs when access to any of the three log files dye list log passwords log or user preferences log has been denied This usually occurs when a user without administrator access runs the software If this is the case someone with administrative privileges must set the permissions on the operating program files folder C ND 3300 Data Log Files so that all users have read write access to the files in the folder To set the permissions do the following 1 Log in using an account with administrator rights 2 Right click on the 3300 Data Log Files folder 3 Select Sharing and Security or Sharing 4 Set the security according to your operating system networking as shown below XP Professional using Dom
51. tatus amp Nema Method Type Source Gun Mn Graph Analysis Require Sid Goin Min nim nm Blonk Vets Cure PicoGreen dsDNA Nutiec Blue 0 500 7 525 TRUE mami F boGreen RNA Blue 9285 TRUE Quant Prowin Protein Blue Auto 0 50 7 600 TRUE wami Poly 3 Linhedternne Omer Uv Auto 0 00 3 460 TRUE t Alexab55 Obo Whe 000 500 700 565 TRUE iep Aluxab47 Other Whe Auto 0 550 750 665 TRUE n Fluorescein FITO FAM Other Blue Auto 0 so 515 TRUE Queene sukata Ote Uv Q 400 450 TRUE wami interp EviTag UV Ote w 612 TRUE ugiml erp Other White Auto 560 7 515 TRUE nM Note prodelrmod methods are indicated with a diamond and cannot be mod ed To add a new method highlight a method and click on Insert Below A new window will appear with several tab options Edit method parameters Edit method parameters Method name Method name Alexabbb Fluorescence Analysis General Fluorescence Analysis Source Correction Type lt Other Source White i 4 Graph Min nm 480 AutoGain pq 4 Graph Max 700 Minimum Gain 000 General tab allows the user to
52. tical fiber and the reduction in scattering from directly wetted optical surfaces An additional element making this method possible is the high reproducibility of the relative spectral output intensity vs the wavelength of the LED In practice a Virtual Emission Filtering Interval is set for each Method created that uses the White LED See section 4 for details regarding the AX Operation A 1 2 ul sample is pipetted onto the end of the lower measurement pedestal the receiving fiber A non reflective bushing attached to the arm is then Section 1 Overview brought into contact with the liquid sample causing the liquid to bridge the gap between it and the receiving fiber The gap or pathlength is controlled to 1mm Following excitation with one of the three LEDs emitted light from the sample passing through the receiving fiber is captured by the spectrometer The instrument is controlled by NanoDrop fluorospectrometer software run from a PC where all data is logged and archived in the C ND 3300 Data folder as well as at a second user defined location An external power supply is not required The operating power is supplied by the USB port on the PC Applications Fluorescence measurements are simple for samples as small as 1 2 ul using the ND 3300 The small sample requirement and ease of use make it ideally suited for fluorescence measurement of a variety of dyes and fluorophores The image below lists some of the most common
53. tion WARNING The system software must be loaded onto the PC before the USB cable is connected Administrator access on the PC is required to install the software To properly install NanoDrop software 1 Close all programs and make sure that the USB cable is unplugged 2 Insert the operating software CD in the CD drive of the PC The software installation menu should appear automatically software menu does not appear choose My Computer to view the contents of the CD Double click on the file named nd3300 version install exe 3 After software installation remove the sticker on the back of the instrument and connect the USB cable The Found New Hardware Wizard should start as shown below Windows XP SP2 operating system will ask to allow it to search the internet for the proper software as shown Select No not this time Follow the prompts for automatic installation of the software Welcome to the Found New Hardware Wizard talado wall seach bx curari ep dit ollo ha air on pour on ihe hacen rad abaco LO gon jur compas dus radeon oda Wirt pmi end CER VUE Y hades came PE an rial LL 2 ope moil Al rar Fite aly cones Intro Page Windows XP SP2 All Windows Operating Systems Your NanoDrop ND 3300 Fluorospectrometer should now be ready for operation If the software does not start properly refer to the Tr
54. u MEL Lu RM LI M 4 5 Show Context toe sisse EA 4 6 Users Mariali citet e tdo 4 6 Mri 5 1 93259 TIOCCIS I irse catio qu taa aU 5 1 dsDNA PicoGreen dye 5 1 RNA RiboGreen dye seen 5 2 dd y a 6 1 TOME Samples ai aa 7 1 Fluorescein Mts 7 1 Alexa Fl or 99 Oil iia ica 7 1 5y5 Alexa cle dee Tode totis ts doct 7 2 uitio SUIT ALS ada 7 2 4 Methyl umbelliferone 4 7 2 8 Archived Data and Data Viewer eere eren nennen nnn 8 1 Archive File Er IN tae 8 1 Data Storage Elierarehiy det Can Uo e run v EUR LY dis 8 1 Dala VIEW a ed dee n 8 1 Opening Archived Data with spreadsheet programs 8 5 9 TFOUDIESHOOUING ici 9 1 EMO FUSB2000 anna 9 1 Gonnec IO MENOS iaa 9 2 ERO OCS Oo X cc 9 3 NStale nero D 9 3 Other Software Error Messages ccccccooconccconccocononnncnnnncnonnonnnnnnnnnonnnannnnnns 9 3 Sampling 9 4 Unusual SPCC A 9 5 Technical SEI EET CH 9 5 10 Maintenance and 10 1 A A esate wer ey al 10 1 Decontamination of Measurement amp Optical Surfaces
55. ually adjusted by 20 nm of the analysis wavelength within the measurement module Note 2 An equation formula comprised of up to four wavelengths and four coefficients can be defined and will report both the RFU of the respective wavelengths and the relationship amongst them The formula is used to calculate concentration See section 3 General Operation for further detail Note 3 All fluorescence measurements outside the virtual filter interval are set to zero The default interval is 20nm and can be set using the User Preferences Table see below Note 4 The AX can also be modified within the respective fluorescence method module Data Viewer This module is designed to offer the user the ability to customize report structures import archived data and re plot previously archived data See section 8 for further details Users Manual A pdf version of the user s manual is available via the Main Menu User Preferences Each user can select settings for two common features used for application modules The default setting for saving data in a report is for Auto Report to be enabled The user may elect to deselect this option using the Change Settings button The same button also allows the user to select a previously saved report column configuration as the default for particular methods This feature is only available when there are stored alternate report formats to choose from T Leer Preferences cor Defmult Saw
56. ully Software Architecture and Features Main Menu With the sampling arm in the down position start the NanoDrop software by selecting the following path Start gt Programs gt NanoDrop gt ND 3300 version NanoDrop ND 3300 2 5 0 File Help User Default Nucleic Method User Acid Editor Preferences Data Account Viewer Management Other Change Samples Password Application Modules 3 2 Section 3 General Operation The software has been tailored to meet the life scientist s needs It includes the following pre coded application modules e Nucleic Acid using 33258 Hoechst dye PicoGreen RiboGreen e Proteins using Quant iT Protein assay e Other Samples includes FITC fluorescein methods for using either the Blue or White LED Cy3 Alexa 555 and 5 647 4 methyl umbelliferone and Quinine sulfate Method Editor The Method Editor button accessible from the Main Menu gives the user the ability to include or configure new fluorescent dyes and methods adding to those currently available Method Name The Method is the unique name used for both inherent fluorophores e g FITC and named applications e g PicoGreen Five 5 buttons at the bottom of the Method List in the Method Editor are used to configure change remove and save new methods Note Diamonds indicate predefined methods which cannot be modified see figure below Method Editor Method List Sort by Protected s
57. wavelength of the spectrum including the fluorophore of interest Sample ID The Sample ID is highlighted for overtyping or barcode scanning The user may input a sample ID that will be used to identify the measurement in a report print and in the archived data file The sample ID entry is key focused meaning it is the default selection on the screen and should have a flashing text cursor when the instrument is waiting to make a new measurement Note The sample ID name can be changed only by using the Data dropdown box on the reports page ND 3300 Data Viewer File Configuration 8 Reports Help Import Samples Ctrl I Plots Rep Rename Selected Ctrl M Delete Selected Ctrl D Delete All Samples Choosing Rename Selected will bring up the following dialog box Rename Sample ID Rename the Sample ID of the selected sample and click Change or Cancel Sample 10 test Note the sample ID in he archive file will also be changed Source specifies the excitation source for the method selected Note The source cannot be changed for predefined methods RFU at xxx nm This is the relative fluorescence output at the wavelength specified for the method user settable cursor The user may move the cursor 20 nm of the analysis wavelength 4 4 Section 4 Common Module Functions For White LED excitation the virtual emission filter interval is the respective wavelength range over wh
58. wer via Show Report Tool Bar Features common to all three pages include Import Configuration Options controlled by this tool bar function include Auto Scale Include graph in printout and Include standards in printout Data Includes options to import data rename samples and delete sample data Note After deleting all samples it is important to exit out of the Data Viewer module and re enter if importing data for a different application type Report This tool bar function allows the user to select columns of interest to be included in a report See section on Reports Page for more detail File Page Set up Allows the user to define the page set up for printing out the spectra the report and the standard curve Print Window The current Plot Report or Standards screen may be printed by selecting Print Window or CTL P Save Window Saves files as jpgs Help Context Help This feature is enabled in the Main Menu all function modules and the application modules The help feature is enabled by choosing Show Context Help from the Help menu pull down or by selecting Ctrl H Once enabled placing the cursor on elements of the screen will automatically generate an explanation of that element Context Help remains active until the user deselects it Accessing the Data Viewer via the main menu requires the user to import archived data Use the Data drop down tool on the top menu bar to select Import Sam
59. y3 Alexa555 Source White 4 1 Section 4 Common Module Functions Print Screen F4 The Print Screen button will print a copy of the current operating screen to the default printer attached to the operating PC Print Window A Print dialogue can be initiated from the File pull down menu or by typing Ctrl P The user can specify which networked printer to use from the Print dialogue Print Report F5 Selecting the Print Report F5 button will print the existing sample report to the default printer It does not clear the sample report contents The default maximum buffer capacity is 32 samples although the user has the option of increasing the buffer to a maximum of 200 samples The user also has options as to how the buffer is handled automatically clear upon printing prompt before clearing etc See section 8 Data Viewer for additional details All data is stored in the archive file at C ND 3300 Data Saving Current Screen as jpg Image The current screen can be saved as a jpg image file by selecting Save Window from the File pull down menu Recording F6 The default setting has the Recording feature activated which automatically records and prints 32 measurements in a report format See section 8 Data Viewer for additional details Note To override this feature click on the Recording button Once de selected the button will read Start Report Print and Save Menu A print dialogue can be initiated fr
Download Pdf Manuals
Related Search