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NanoDrop ND-1000 Spectrophotometer manual

1. 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 16 1 Connection Error 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 If your PC is automatically going into standby or hibernate mode the USB communication will be lost whenever it occurs and Retry will NOT reconnect the instrument If this occurs the USB cable will need to be disconnected 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 The System Standby and System Hibernate should be set to never for the Plugged In column as s
2. Directory Tree i T j j 7 j d 8 ef mmm nu An so Si si Weer ere Lanier DO i a Eet do eN appii A acio a Pry ll m d i be Gore Launch Arches Fie Cor rer K feg emake Pa eet A es 20 E Select Loge Deseloct akot Search Gosrch la rapier to Balen current ralk brant ol Sep Pe hi Plots The Plots page is where selected sample spectra are displayed Tes type Muchaic Acid LAILU waali la Selected plot ders aingi Dote gi Sample nicemator des 40l sgid Lata 10 26 5006 Th aed KL Wer erie coe ips A 1 ee Plots Features include Test Type Auio fills in module name Date Auto fills in the date and time when a report is generated Selected Plot There are two methods of selecting or highlighting individual sample data The 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 14 3 Section 14 Archived Data and Data Viewer Plots Sets Users may select the maximum number of individual plots up to 20 graphed per page Since a report can hold data for many 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
3. ran pepon i ShowPepon eg Cintas DR 5311 50 00 45 00 Sample iD ds Ci 40 00 a 35 DI S 3000 Sample 16 2 25 00 e ki 230 mm Abs 10452 20 00 r z 15 00 A A26010 men pe 23714 3 P x 1000 e oo ACEO 10 een path 1001 5080 1 80 ES 227 0000 531 D i i i E E E D i i i D i 220 230 240 250 260 270 280 250 300 30 320 330 340 I0 W pn i ncb nm ngul 11857 151 0 06 00 OG The default option is set to clear the display for the next reading The user may set the overlay control to clear after each sample plot default setting after each new report or accumulate plots until prompted to clear The Clear Now setting will clear all current and previous plots When the overlay function is active the software will auto scale the y axis based on the sample with the highest absorbance at 260 nm Note When the overlay function is active the Blank function does not clear the existing overlaid sample spectra 9 2 Section 6 MicroArray 6 MicroArray The capability to pre select viable fluorescent tagged hybridization probes for gene expression in micro arrays can eliminate potentially flawed samples and improve research effectiveness The NanoDrop ND 1000 Spectrophotometer measures the absorbance of the fluorescent dye allowing detection at dye concentrations as low as 0 2 picomole per microliter Fluorescent Dye Selection There are currently nine fluorescent dyes
4. CONTINUE CANCEL 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 c nanodrop data log files folder It is strongly recommended that the administrator make a copy of that file and store it in the same log files folder as above each time a new user account is added or a password is changed If the administrator s account becomes locked the up to date copy can be renamed and used as the password log file Note If upgrading from a previous version the passwords log and user preferences log files should be automatically copied to the c NanoDrop DatalLog Files directory If for some reason these files are not copied automatically they must be manually copied from the c program files NanoDrop version to the c program files NanoDrop version directory 3 5 Section 3 General Operation Dye Chromophore Editor The Dye Chromophore Editor gives the user the ability to add their own dyes or chromophores in addition to the predefined fluorescent dyes available for use with the MicroArray and Proteins and Labels modules Note 1 Predefined dye methods are indicated by a diamond and can t be modified Note 2 Absorbance contribution at 260 nm and 280 nm from the respective dye can be corrected by entering the appropriate decimal correction field Refer to the dye manufacturer to find the 260 nm and 280 nm factor for dyes not pre defined
5. Archive File Creation Every time an application module is started an application specific archive file is created for the user that is logged in All measurements made by the user in that application module 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 Nucleic Acid 2007 06 04 ndj corresponds to Nucleic Acid data from the software session that began on June 4 2007 A unique file extension ndj has been given to these files to enable automatic startup with the Data Viewer see section on Data Viewer below Mame Size Type Date Modified z Nucleic Acid 2007 06 13 3 5 ndj d KB NDI File 6113 2007 7 53 AM sl Nucleic Acid 2007 06 04 3 5 ndj d KB NDI File 6 4 2007 4 00 PM 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 Absorbance data shown in archive files are represented as they are displayed on the screen For Nucleic Acids Protein A280 and Protein and Labels application modules data are stored based on a 1 0 cm 10 0 mm path For MicroArray UV Vis BCA Protein Bradford Lowry and Cell Culture application modules the data are normalized to a 1 0 mm 0 1 cm path For high absorbance UV Vis samples data are stored based on a 0 1 mm path Note 2 For data from
6. Wavelength The wavelength is auto calibrated based on known peaks in the xenon lamp spectra each time the software is started No calibration is required by the user Pathlength Accuracy Generally this is not required as field experience has shown that the path does not change appreciably even after several years of heavy use However it is good practice to verify the calibration every six months using CF 1 calibration fluid The calibration check procedure is found within the Utilities and Diagnostics module Parts That Require Replacement In general the only part that should periodically require replacement is the flash lamp The flash lamp should last a minimum of 30K measurements There is no way to test the flash lamp to confirm its remaining life When the flash lamp fails the light output will become very erratic or stop altogether Warranty All spectrophotometers and accessories manufactured by NanoDrop Technologies are warranted against manufacturing defects in parts and labor for a period of one year Preventive Maintenance as well as additional one two and three year warranty extensions are available Plan descriptions can be found at the following link www nanodrop com 17 1 Section 18 Appendices 18 Appendices Instrument Specifications e Sample Size 1 microliter e Path Length 1 mm with auto ranging to 0 2 mm Light Source Xenon flash lamp Detector Type 2048 element linear silicon CCD array Wavelength Rang
7. 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 idVendor 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 Intel r 82801DB DBM USB Universal Host Controller 2402 bedUSB gt RootHub bDeviceClass 3 bDeviceSubClass Port NoDeviceConnected bDeviceProtocal Port2 NoDeviceConnected agRacketSizel wel SA GA Intel r 82801DB DBM USB Universal Host Controller 2404 idVendor S gt RootHub idProduct i Port1 NoDeviceConnected EE EE Dages Port2 NoDeviceConnected iProduct Intel r 82801DB DBM USB Universal Host Controller 24C7 iSerialNumber RootHub bNumConfigurations 0x01 Port NoDeviceConnected ConnectionStatus DeviceConnected Port2 NoDeviceConnected Current Config Value 0x01 Intel r 82801DB DBM USB 2 0 Enhanced Host Controller 240D Device Bus Speed Ful RootHub Device Address Open Pipes Port1 NoDeviceConnected Port2 NoDeviceConnected Endpoint Descriptor Port3 NoDeviceConnected i Port4 NoDeviceConnected Port5 NoDeviceConnected Port6 DeviceConnected Generic USB Hub Port1 NoDeviceConnected Endpoint Descriptor bEndpointAddress 0x00 Port3 NoDeviceConnected Transfer Type Control ee HoDeviceComriestad wMaxPacketSize Ox0507 1287 bInterval
8. e CD ROM drive e 32 MB or more of RAM e 40 MB of free hard disk space e Open USB port the instrument can only be connected via the USB port e Microsoft Excel or other spreadsheet program to manipulate archived data optional Software Installation 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 If the software menu does not appear choose My Computer to view the contents of the CD Double click on the file named nd 1000 install exe 3 After software installation connect the USB cable and 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 Hew Hardware Wizard SE E PEP E EE sch kw duei i att oni hes hahae mal ahi Siew aisen LO on H foo Joie wett ge elt ye peer he Skeet Klees Desiree poy E Cer wh an intro Page Windows XP SP2 All Windows Operating Systems Your NanoDrop ND 1000 Spectrophotometer should now be ready for operation If th
9. pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm See 260 280 Ratio section of the Troubleshooting section for more details on factors that can affect this ratio 260 230 ratio of sample absorbance at 260 and 230 nm This is a secondary measure of nucleic acid purity The 260 230 values for pure nucleic acid are often higher than the respective 260 280 values They are commonly in the range of 1 8 2 2 If the ratio is appreciably lower this may indicate the presence of co purified contaminants ng ul sample concentration in ng ul based on absorbance at 260 nm and the selected analysis constant See the Concentration Calculation Beer s Law in the appendix for more details on this calculation Spectrum Normalization The baseline is automatically set to the absorbance value of the sample at 340 nm which should be very nearly zero absorbance All spectra are referenced off of this Zero Spectrum Overlay Control The user can display more than one spectrum in the same display using this feature The current sample plot will be displayed in bold and previous plots will be distinguished by different colors as seen in the following example Aipclete Acids rie Echt Help Rua blank Prat Screen Reponn Mentuzermest completa 67492007 1 30 PM Bien
10. unknown Making Lowry Measurements A standard curve is required every time the Modified Lowry assay is run Although curves can be saved and reloaded in the NanoDrop ND 1000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay Additionally a standard curve set up may be reloaded This feature will recall the respective standard series used in a previously saved standard curve Both single and multi point standard curve generation is incorporated into the software A standard curve can be developed using a reference Modified Lowry reagent only no protein and a single replicate of one standard The multi point standard curve generator allows a maximum of five replicates for each of seven different standards There is no set order in which standards must be run A blank must be measured before the standard curve may be generated It is advisable to use water as the blank and use the dye reagent without any protein added as the 0 or reference sample There are three general procedural steps to unknown protein concentration measurements The requisite order including generating the standard curve is as follows Step 1 Measure the Reference Lowry reagent a zero Standard Note The software will guide the user with instructions in the large text box on the right side of the screen 11 2 Step 2 Measure Stan
11. Acids The default setting is DNA 50 Other options include RNA 40 ssDNA 33 and Other with a variable constant setting e UV Vis The default settings for the two cursors used to monitor specific wavelengths are 300 nm for 11 and 700 nm for A2 The user may elect to have the HiAbs on automatic utilization of the 0 2 mm path An additional option is to elect to 3 3 Section 3 General Operation normalize the data and spectra using the absorbance value of the wavelength between 400 nm and 700 with the lowest absorbance value e Microarray The default setting is ssDNA 33 for the nucleic acid The default setting from NanoDrop remain Dye 1 set to Cy3 with absorbance normalized to the absorbance value at 750nm Other options include RNA 40 ssDNA 33 Other with several hard coded dye choices including common Alexa fluor dyes See the section on Dye Chromophore Editor at the end of chapter 4 for more information e A280 There are six sample types options available for purified protein analysis and concentration measurement The default setting is Other protein E1 See Section 8 for additional information about each sample type option Note New to version 3 5 1 is the ability to select whether or not to have the data and spectrum normalized to the absorbance value at 340 nm e Proteins and Labels There are six sample types options available for purified protein analysis and concentration measurement The default setting is Other protein
12. E1 The user may also use elect whether or not to use a bichromatic normalization to the absorbance value at 340 nm The default setting from NanoDrop remains Dye 1 set to Cy3 with absorbance normalized at 750nm Utilities and Diagnostics This module is used to both confirm that the instrument is performing within the pathlength calibration specifications and help troubleshoot operational problems with the instrument Note The inclusion of the calibration check utility is new to version 3 5 1 For more information on using this module refer to both section 15 Calibration Check and section 16 Troubleshooting of this manual Account Management The Account Management module provides options for directing where specific data files are archived by allowing users to segregate their data into personal folders The Account Management module is accessible to the administrator only Account Types There are three types of user accounts e Level 10 this is the highest security setting and all level 10 users 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 nanodrop It is strongly recommended that the password be changed after initial account set up Any user can be set to a level 10 access although this is not recommended see Level 5 Note The administrator or the last level 10 user account may not be dele
13. Spreadsheet Programs The archived files are in tab delimited format and can be opened in Microsoft Excel or an equivalent spreadsheet program When opening with Excel be sure to save the file as a new name before making any changes to the data If changes are made to the data in the ndj file the Data Viewer module may not be able to recognize and import the data 14 6 Section 15 Calibration Check 15 Calibration Check The Calibration Check is found within the Utilities and Diagnostics module and is accessed through the Main Menu It is used to confirm that both pathlengths are within calibration specifications Utilities amp Diagnostics ees Diagnostics nostics Es wgl to a PETN wgl Menu A vial of CF 1 is required to run the calibration check procedure CF 1 is an aqueous potassium dichromate K2Cr207 solution for use in confirming calibration of NanoDrop Spectrophotometers It is roughly ten times more concentrated than other commercially available K2Cr2O7 solutions used to confirm calibration of standard spectrophotometers and is available through NanoDrop technologies or your local distributor Procedure Ensure the measurement pedestals are clean and that a 1ul water sample beads up on the lower pedestal 1 Open the ND 1000 Calibration Check Software and follow the prompts in the Customer Guidance text box of the software 2 Enter the Target Absorbance found on the CF 1 vial as directed in the image below
14. The Delete Point button allows the user to delete points by first selecting the data point to be deleted in the table and then choosing the Delete Point button If erroneous or non representative readings are encountered for a specific standard all replicates of that standard are cleared by selecting Delete 1 Standard Additionally all standards can be deleted at once using the Reset all Standards button User Defaut momi AveAbs Abs 1 2 Abs 3 Abs4 Abs 5 6 4 2007 443 FM 0009 1000 9 009 0001 0209 0001 G 000 0 090 1514 0345 0 11 48 54 050 0016 6016 ope LA gt Siendord 3 0759 00 0605 oms Sienderd4 1000 003 003 0 033 SrenderdS 3000 0107 Gun O15 Sadari 4000 0146 0145 oww SE Modified Lowry Kr Standard Curve 0 2 4 0 mg ml Mirtha aida eat 0 00 O20 049 050 080 100 129 140 160 189 200 220 240 269 280 ogi Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module Section 12 Protein Bradford 12 Protein Bradford The Bradford Assay is a second alternative method commonly utilized for determining protein concentration It is often used for more dilute protein solutions where lower detection sensitivity is needed and or in the presence of components that also have significant UV 280 nm absorbance Like the BCA and Lowry methods the Bradford assay requires a standard curve
15. 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 Plots Report Standards Testtype Lawy S SAUC TANAN La Report Name Max Buffer Size 200 Buffer Mode save Report amp Clear Y Sample Date Time 1 ngful A260 A280 260 280 260 230 Constant 2 Cursor Cursor 340 Measurement Al ID Pos abs raw Type Data Storage Hierarchy The hierarchy for archive files is as follows C NanoDrop Data gt User name gt Application Module BCA Protein Lowry Bradford Cell Culture MicroArray Nucleic Acid Protein A 280 Proteins amp Labels UV Vis or UV Vis HiAbs All archived data files are stored within an application module folder that is within the User folder as shown below Be C NanoDrop Data Default 3 10 x File Edit View Favorites Tools Help ae Back gt i S 4 2 Search Folders gt 7 A iZ EI Address la C NanoDrop DatalDefault sl E EI Go Norton Antivirus a Folders x mame Le swe tpe ateModfied gt E Documents and Setti
16. amp NanoDrop ND 1000 Spectrophotometer V3 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 is a registered trademark of NanoDrop Technologies Inc Other parties trademarks are the property of their respective owners and should be treated as such Copyright 2007 NanoDrop Technologies Inc rev 7 2007 Table of Contents OVERVICW EE 1 1 INStUIMENTDESCH DUO WEE 1 1 Operaio Misas in 1 1 eieiei 1 1 SE 1 1 JEE d Py Ss ce eee Se ee Sk 2 1 Computer Requirements oooccccccccccconnnccnnncnononononnnnnonononnncnnnnnnonnnnnnnnnnnnns 2 1 Software MS cia 2 1 Software Neier 2 2 Registering Your Instrument 2 2 General OPC ration sicciciirrici ii ias 3 1 The Sample Retention Gvstem 3 1 Cleaning the Sample Retention Gvstem AAA 3 1 Software Architecture and Features 3 2 o tere 3 3 Utilities and DANOS o atte ad secs 3 4 Account Management 3 4 Dye Chromopnore ele EE 3 6 Common Module Functions cccccccssssseseeeeeecccessseeeeeeeeeensseeeensseees 4 1 Module e e EE 4 1 Common FUNCIONS dE 4 1 Measures ic llos 4 1 Blank F A R een dade uataeausaeees 4 1 o R2 e e aa a a r 4 2 lee e RR EE 4 2 Stan REDO RECORGING EE 4 2 PrN Re elle EE 4 3 SNOW REPOM EE 4 3 Sample RE 4 3 EE 4 3 EE E ee ewce as eae etece 4 3 EsCape Key ESC cities ege Radeon teed a
17. 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 is automatically saved to an archive file and requires no user action Escape Key ESC The escape key is set to exit out of all screens Hitting the escape key twice will log the user out of an application module Show Context Help Ctrl H Context Help 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 deselected User s Manual A PDF version of this User s Manual is accessible from the Main Menu and from the Help menu in all of the application modules 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 1000 version 4 3 Section 5 Nucleic Acids 5 Nucleic Acids Nucleic acid samples can be readily checked for concentration and quality using the NanoDrop ND 1000 Spectrophotometer To measure nucleic acid samples select the Nucleic Acid application module Nucheke Acida rie Edi Help Eu blank Pret Screen Recoma HMes
18. blank must first be measured before the Measure button will become active The Measure button is used to initiate the measurement sequence for all samples non blanks It is actuated by depressing the F1 key or clicking the Measure button The entire measurement cycle takes approximately 10 seconds Blank F3 Before making a sample measurement a blank must be measured and stored see Blanking and Absorbance Calculations in the appendix for more details on absorbance calculations After making an initial blank measurement a straight line will appear on the screen subsequent blanks will clear any sample spectrum and display a straight line as shown in the following image T Achebe Acids rie Edi Holp BReblank Prnt Screen bake naw BLAME 6752007 9 36 AM 3 gcorbag e Y g O Uren d Bank runpepon ShowReport Reeder Outaht Chile cone Clear graph esch Semple Sample Type AMS Sample 1D Sampler 0 ki 210 im Aba 0 000 Ao min palh Nah 28010 mm path Hab Pepa Mam E Neh dis D i i i i a D i i i 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wien length pm ngul NaN 251 0 D542 ea For the most consistent results it is best to begin any measurement session with a blanking cycle This will assure the user that the instrument is working well and that the pedestal is clean To perform a blanking cycle perform the following 1 Load a blank sample the buffer solvent or car
19. concentrations can be calculated by Sample Sandars l using linear e e interpolation point to point between the two standards flanking the unknown sample or by using polynomial EE fitting Note In order POR to obtain a concentration value mg ml the sample Abimi Sn UTH 575 0 i me n rs ugiml 1174 PERE J f t ote 5 J unknown must fall within the limits of the standard curve Standard Curve Features Standard curves can be saved and reloaded for reference use by using the Standard Curve pull down menu and choosing the Save as or Load Standard Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time New to version 3 5 1 is the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete Standard Points The Delete Point button allows the user to delete points by first selecting the data point to be deleted in the table and then choosing the Delete Point button If erroneous or non representative readings are encountered for a specific standard all replicates of that standard are cleared by selecting Delete 1 Standard Additionally all standards can be deleted at once using the Reset all Standards button 12 3 User Default Abs 5 16 12 2007 2 07 PM 0022 x dE ISLAMIS 3 BAR 0067 User Ip wein Abs 5 16 12 2007 207 EM 00 0041 00
20. curve concentration series using the Load Standard Curve Set up Delete Standard Points The Delete Point button allows the user to delete points by first selecting the data point to be deleted in the table and then choosing the Delete Point button If erroneous or non representative readings are encountered for a specific standard all replicates of that standard are cleared by selecting Delete 1 Standard Additionally all standards can be deleted at once using the Reset all Standards button 10 3 User Detaun faso SIAM 9 008 000 0008 0003 i ams 0003 000 ISLA DIAS 0 11 64 24 om 003 ons 0081 D 0152 10279 ETT 3 I m g 9 ER ES GI S ES 87 A e Ep y 4 4 b 84 eoe A uw ap Regular BCA Standard Curve 0 2 8 0 mg ml User Detaua ni REOSE EE 0003 0004 0003 d d 6010 0010 ji i i 1914 112545 0084 24 awe 0027 0065 0065 0146 014 0276 0279 dad ual ad eg ed i 0 01 0 20 mg ml Exiting the BCA Module mini BCA Standard Curve Section 10 Protein BCA It is recommended that you process all of the unknowns to be assayed before exiting the BCA software module 10 4 SE 11 Protein Lowry The Modified Lowry Protein Assay is an alternative method for determining protein concentration based on the widely used and cited Lowry pro
21. help from your distributor or NanoDrop Technologies Saturated Detector The detector is saturated This is most likely caused by initialization with dirty measurement pedestals IF the error persists clean the pedestals exit the software and restart This error message can occur when the software calculates too high integration times during initialization This is most likely due to a dried sample left on the measurement surface after the last use of the instrument Cleaning both the top and bottom pedestals with de ionized water and exiting out of the software module to the main menu should alleviate the problem It is not necessary to close the software completely as each module is re initialized when it is opened If the error persists contact NanoDrop Technologies or your local distributor Liquid Column Breakage 16 3 Warning The last measured absorbance 0 625 exhibited an error greater than 10 This might be caused by 1 An air bubble in the sample 2 The liquid column is not forming can be confirmed visually If this is occurring try cleaning both measurement pedestals refer to cleaning procedure by clicking on button below If column breakage persists use a larger sample size 1 5 2 ul 3 One or both of the measurement paths is out of calibration Ifthis error persists and the sample column is forming properly contact NanoDrop Technologies or your local distributor As a result of this error C
22. in the Dye Chromophore List Note If upgrading from a version prior to 3 3 zero values 0 for 260 nm and 280 nm correction factors will be entered for all user defined dyes Dye Chromophore List Editor Dye Chromophore List Name Alexa Fluor 488 Alexa Fluor 546 1 04E 5 Alexa Fluor 555 1 50E 5 Alexa Fluor 594 7 30E 4 Alexa Fluor 64 2 39E 5 Alexa Fluor 660 1 32E 5 4 0y3 5 1 50E 5 4 0y5 5 2 50E 5 Note predefined dyes are indicated with a diamond and cannot be modified 3 6 Section 4 Common Module Functions 4 Common Module Functions Module Startup When the software starts you should see this message Ensure sample pedestals are clean and then load a water sample After loading water sample click OK to initialize instrument For best results ensure measurement pedestal surfaces are clean and load a water sample onto the lower measurement pedestal and then click OK After clicking OK the message Initializing Spectrometer please wait will appear When this message disappears the instrument will be ready for use All data taken will automatically be logged in the appropriate archive file Common Functions Eile Edit Help Make new BLANK 6 4 2007 4 00 PM Measurement Exit Default Measure F1 Each time a software module is opened initiated the Measure button is inactive as noted by its grayed out appearance A
23. install the software without administrator privileges Contact your system administrator to install the software Source Error This error indicates that there is insufficient light getting through to make good absorbance measurements Check that the sampling arm is in the down position and the power is connected Error 9003 This error indicates that the monitor resolution is below the 1024x768 required Check the computer settings Be sure that the start menu tool bar is set to the bottom and not along the side Low Detector Bias This occurs when the software has detected a problem with the detector Contact NanoDrop Technologies or your distributor if you encounter this error OOIDRV Timeout This error occurs when a necessary file is deleted as a previous version of the software is uninstalled Reinstall the newest software to correct the problem EZUSB SYS Cannot Be Found If this error message appears do the following 16 5 e Windows 2000 Type C WINNT INF in the file path text box e Windows XP Type C WINDOWSIINF in the file path text box This should allow the installation to complete successfully Driver X Configuration Failed You Must Manually Edit the Registry This error message or others with similar wording occurs when attempting to install the operating software ona computer running Windows 2000 or XP It occurs because the user does not have the necessary authorization to install the software Contac
24. linear CCD array is used to analyze the light after passing through the sample The instrument is controlled by special software run from a PC and the data is logged in an archive file on the PC Applications UV VIS spectrophotometry is simple for samples as small as 1 ul using the NanoDrop ND 1000 Spectrophotometer The small sample requirement and ease of use make the NanoDrop ND 1000 Spectrophotometer ideally suited for measuring e Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng ul dsDNA without dilution Fluorescent dye labeling density of nucleic acid microarray samples Purified protein analysis A280 up to 100 mg ml BSA Expanded spectrum measurement and quantitation of fluorescent dye labeled proteins conjugates and metalloproteins Bradford Assay analysis of protein BCA Assay analysis of protein Lowry Assay analysis of protein Cell density measurements e General UV Vis spectrophotometry Patents The sample retention technology used in the ND 1000 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 e Microsoft Windows XP or 2000 operating system Windows Vista has also been tested successfully with NanoDrop software The operating software is not compatible with Windows NT 95 98 or ME e 233 MHz or higher processor
25. of 1 8 and 2 0 for DNA and RNA are rules of thumb The actual ratio will depend on the composition of the nucleic acid Note RNA will typically have a higher 260 280 ratio due to the higher ratio of Uracil compared to that of Thymine 8 Leninger A L Biochemistry oe ed Worth Publishers New York 1975 Unusual Spectrum A sample that exhibits jagged cuts out of the spectrum but an otherwise normal shape may be the result of detector saturation This can be caused by the software selecting too high of an integration time due to a dirty sample pedestal upon startup Try cleaning both sample pedestals thoroughly and restarting the software For reference examples of spectra generated with a saturated detector are shown below 16 7 SE e Detector saturation nucleic acid Detector saturation Bradford measurement measurement A spectrum that is very un smooth or ragged can be caused by insufficient light intensity reaching the spectrometer If you suspect that this is occurring refer to the Technical Service section for instructions on how to contact NanoDrop Technologies and what information must be sent to allow for diagnosing the problem 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 at info nanodrop com for help The following information will be very helpful e Serial Number of
26. surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer the Modified Lowry assay can run from 0 20 mg ml up to 8 0 mg ml Assa Approx Approx Typical Reproducibility 7 el Lower Upper minimum 5 replicates yp Limit Limit CV Modified Lowry 0 2 mg ml 4 0 mg ml 2 over entire range Modified Lowry Kits Protocols and Sample Preparation Commercial Modified Lowry Protein kit manufacturers typically outline procedures for their assays Modified Lowry assay using a 5 1 reagent sample volume ratio e To accurately prepare Standards we suggest using a minimum sample volume of 20 ul and 100ul of Modified Lowry reagent Using the same pipettor for both volumes will eliminate any pipette to pipette accuracy differences e After the initial 10 minute room temperature RT incubation period add 10 ul of the Folin Ciocalteu reagent and incubate for 30 minutes at RT In addition to the kit reagents protein standards BSA for generating a standard curve are also provided by the manu
27. the PC Print Window A Print dialogue can be initiated from the File pull down menu or by typing Ctrl P The user can specify any connected printer to print to from the Print dialogue 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 Start Report Recording The user can log measurement results in a report table and print them to the desired printer To initiate this feature select the Start Report button The default setting has the Recording feature activated Refer to section 14 Data Viewer for additional details Note To override this feature click on the Recording button Once de selected the button will read Start Report When the specified maximum number of entries for that specific report has been reached there are 4 options Ignore Save Print Save and Print All data is stored in the archive file at c WanoDrop Data and in a duplicate location if selected in User Preferences 4 2 Section 4 Common Module Functions Note This feature can be set so that Recording is the default mode See User Preferences in section 3 for more information Print Report F5 Selecting the Print Report F5 button will print the existing sample report to the default printer It can be configured to clear the sample report contents The user also has options as to h
28. visual indication of the operability of the 12V power supply 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 updates 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 Basic Use The main steps for using the sample retention system are listed below a sampling 1 With the sampling arm open pipette the sample onto the lower measurement pedestal 2 Close the sampling arm and a initiate a spectral measurement nen Using the operating software on the PC The sample column is automatically drawn between the upper and lower measurement pedestals and the spectral measurement made lower measurement pedestal Na 3 When the measurement is complete open the sampling arm and wipe the sample from both the upper and lower pedestals using a soft laboratory wipe Simple wiping prevents sample carryover in Successive measurements for samples varying by more than 1000 fold in concentration See www nanodrop com for performance data on sample carryover Cleaning the Sample Retention System Wiping the sample from both the upper and lower pedestals as shown above upon completion of each sample m
29. 10 10 mg ml 0 10 mg ml l sample range gt 10mg ml 2 Cv3 sample range 0 20 4 0 pmol ul 0 20 pmol ul y 0 2uM 100uM sample range gt 4 0 pmol ul 2 Unique Screen Features Proteins amp Labels Measure M x Absorbance 1100 2 asalini Type in 340 Bichiomalic kloe FSFE O aii d nu HEEN NEE sample Type d oc B 7 00 CU Ge 5 00 sample dl keep Li 260 E Mom Als o 13 1034 ID poup nlopf atinim 3 00 SVE p m a E 5 H H a DN E E E M Dan FTTOEGM bam Abe 4538 3 Il A A Al HI 1 00 E i 1 I i 1 D i i i 1 220250 300 350 am Ap 500 556 600 550 700 50 Mime lencith nm 251A ERD AROS Ab mb aL 11 mim 2 6 Max Absorbance used to rescale the upper limit of the vertical axis Sample Type The same six sample types options listed under Protein A280 Section 8 are available for purified Proteins amp Labels analysis and concentration measurement All of the options can be viewed by clicking the mouse while it is positioned within the sample type box The sample type color keyed can be selected by clicking on the preferred option or by scrolling through the selections using the up or down arrow keys located to the left of the sample type box See Protein A280 Section 8 for a detailed description of each sample type 13 user selected wavelength Norm Abs 13 10mm equivalent normalized absorbance at the respective wavelength Dye 1 or
30. 2 user selected dye Norm Abs 11 normalized 10 mm equivalent absorbance of selected Dye uM concentration based upon selected Dye s extinction coefficient See the Concentration Calculation Beer s Law in the Appendices for more details on this calculation Abs ratio 11 13 ratio of the absorbance of Dye 1 to the absorbance at the user selected wavelength A3 mg ml concentration of proteins in the sample calculated using the absorbance at 280 nm minus the absorbance at 340 nm i e normalized at 340 nm See the Concentration Calculation Beers Law Appendices for more details on this calculation Baseline Type This application module has two user selectable Baseline Type options The default setting is set to normalize the display spectrum at 750nm Alternatively the 400 750 Slope Baseline Type will normalize the display at 750 nm and accommodate any linear baseline offset across the 400 to 750 nm range 9 2 Section 10 Protein BCA 10 Protein BCA The BCA Bicinchoninic Acid Protein Assay is an alternative method for determining protein concentration It is often used for more dilute protein solutions and or in the presence of components that also have significant UV 280 nm absorbance Unlike the Protein A280 method the BCA Assay requires a standard curve to be generated each time it is run before unknown proteins can be measured The resulting Cu BCA chelate formed in the presence of protein is measured at it
31. 50 nm for dye concentration calculations The green vertical line on the screen represents the peak wavelength position for Dye 1 and the red vertical line represents the peak wavelength position for Dye 2 Unique Screen Features I hero array Fie Edi Help d o i 17D 056 Max Absorbance Sample Type soli L sampler IC Mrs sampli Sample d A Hl Abs Hom 015 Ai ad D E 4 o J U d e L P i i 220 750 200 350 40 460 500 550 600 650 700 750 Weselengih pm 351006345 41005616 Max Absorbance used to rescale the upper limit of the vertical axis Sample Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 for RNA ssDNA 33 for single stranded DNA or Other for other nucleic acids The default is ssDNA 33 If other is selected the user can select an analysis constant between 15 150 When navigating amongst the three 3 general sample types within the Micro Array module the last value of the constant entered within the Constant Sample Type will be retained See Concentration Calculation Beer s Law in the appendix for more details on this calculation and Abs Norm the user selected wavelength black cursor and corresponding absorbance at the 1mm pathlength The wavelength can be selected by dragging the black cursor or using the up down arrows in the wavelength box Note The user selected wavelength and abs
32. 67 0104 wa Es ex Stendend Curve Type 2nd Order Polynomial O 35 4 4 50 55 60 065 70 7 80 wn Exiting the Bradford Module 151A 1 FA Regular Bradford curve covers 200 8000 ug ml Note the linear range is 100 1000 ug ml A mini Bradford assay covers an approximate range of 15 100 ug ml Section 12 Protein Bradford It is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module 12 4 Section 13 Cell Cultures 13 Cell Cultures Using an absorbance spectrophotometer to monitor light scattered by non absorbing suspended cells is common practice in life science laboratories Such applications more than any other accentuate the differences amongst the optical systems of the numerous spectrophotometer designs Note The most distinct difference between the NanoDrop ND 1000 spectrophotometer absorbance values for microbial cell cultures and those observed using classical cuvette based systems will be attributable to the shorter pathlength 1 mm vs 1 cm Values may not be exactly 10 fold different as readings are dependent on both the optics of a specific spectrophotometer as well as the cell type in suspension The Cell Cultures module displays the sample spectrum from 250 nm to 700 nm One cursor is fixed at the frequently used wavelength for monitoring cell suspensions 600nm while the second cursor can be set to the wavelength of interest Uniq
33. A Winey tes 10 2 Measuremen Typa ee Serge Esendeeda Fidir rc and Sandan IN 6 064 0012 0 039 A Punters Abbas UN Ababa A US A ce jajaa oe 0 000 5 Step 2 Measure Standards Up to 5 replicates of each of up to 7 standards can be measured The software will not allow measurement of samples until a minimum of 1 standard and references or 2 Section 10 Protein BCA MERA AR ee EOE TIET A E 50 480 500 5 50 560 GI 600 620 440 560 600 20 720 7 misanan Wavsisngh ns SE standards are measured Polynomial curve fitting requires more standard points Step 3 Measure A Samples Sample concentrations can be calculated by using linear interpolation point to point between the two standards flanking the mess 1 unknown sample or i by using polynomial fitting Note In order to obtain a concentration value mg ml the sample unknown must fall within the limits of the standard curve Ababa S cana Diir ET BE 20 460 op 551 Sa 560 5 600 50 440 660 690 200 20 90 mg ml 1 121 IJSS 00016 Wavslngh na Standard Curve Features Standard curves can be saved and reloaded for reference use by using the Standard Curve pull down menu and choosing the Save as or Load Standard Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time New to version 3 5 1 is the ability to reload a standard
34. On the NanoDrop ND 1000 Spectrophotometer the regular BCA assay can measure samples from 0 20 mg ml up to 8 0 mg ml A mini BCA assay covers an approximate range of 0 01 0 20 mg ml esa Approx Approx Typical Reproducibility T a Lower Upper minimum 5 replicates yp Limit Limit SD mg ml CV Regular BCA 0 2 mg ml 8 0 mg ml 2 over entire range Mini BCA 0 01 mg ml 0 20 mg ml 0 01 mg ml over entire range BCA Kits Protocols and Sample Preparation Commercial BCA Protein kit manufacturers typically outline procedures for two different protein concentration ranges e A regular assay using a 20 1 reagent sample volume ratio To accurately prepare standards we suggest using a minimum sample volume of 4 ul in 80 ul of BCA reagent larger sample volume is preferable e Amini assay using a 1 1 reagent sample volume ratio To prepare sufficient volume of these 1 1 mixtures we suggest using a minimum of 10 ul of sample and 10 ul of BCA reagent in a PCR tube Using the same pipettor for both volumes will eliminate any pipette to pipette accuracy differences Note If you run the assay at 60 C doubling the volumes may afford greater insurance against skewed results from evaporation condensation within the sealed reaction tube In addition to the kit reagents protein standards BSA for generating a standard curve are provided for the BCA method by the manufacturer Follow the manufacturer s protocol for the assay
35. Typically the target absorbance is 0 734 the actual value will depend on the lot of CF 1 ND 1000 Calibration Check Customer Guidance gt Measure Enter the target absorban on ial E yb and 1 0 mm path Then add a 1uL 6 4 2007 1 44 PM mess d sel hd Blank E Target Abs 350 nm 1 0 mm path J a asurementn Replicates 0 1 0mm Path 0 2mm Path 0 000 0 00 NaN N aN 1 mm Path bi 0 2mmPath Measured Absorbance vs Target U USU 0 040 3 ZS 0020 A e S e 3 5 2 S 2 E e rA LI E g S oO a 0 000 5 0 020 SH 1 1 1 1 D 1 1 1 2 0 040 220250 300 350 400 450 500 550 600 650 700 750 0 050 Wavelength nm LIRE ee Cao 3 3A152 1 17 48 24 Measurement 3 Add 1ul of deionized water and select Blank 4 Before opening the ampoule of CF 1 Calibration Fluid shake vigorously to ensure solution is thoroughly mixed Ensure all solution is collected in the bottom portion of the ampoule 5 Carefully break the neck of the ampoule to open the CF 1 Calibration Fluid 6 Follow the on screen prompts in the Customer Guidance text box Using individual 1ul aliquots of the CF 1 Calibration Fluid measure 10 replicates After the 10 measurement the calibration check results will be displayed on screen in the Customer Guidance text box 7 If the instrument does not pass the calibration check using 1ul samples immediately repeat the procedure again step 6 usin
36. aaa 18 1 Instrument Specifications oooooncccnnccconoonncnonnnonononnnnnnnnnnonnnnncnnnnnnnnnnnos 18 1 Blanking and Absorbance Calculations ssssssesesennennneeereeeeeesrerrr rr rnnn 18 1 Concentration Calculation Beers L aw 18 1 Solvent Compatibility Decontamination of Measurement Optical Surfaces 000nnn00010eena 18 2 Setting Up a Dymo 400 Label Writer Printer ccccooccccccccnccccncnnnno 18 2 Section 1 Overview 1 Overview Instrument Description The NanoDrop ND 1000 is a full spectrum 220 750nm spectrophotometer that measures 1 ul samples with high accuracy and reproducibility It utilizes a patented sample retention technology that employs surface tension alone to hold the sample in place This eliminates the need for cumbersome cuvettes and other sample containment devices and allows for clean up in seconds In addition the ND 1000 has the capability to measure highly concentrated samples without dilution 50X higher concentration than the samples measured by a standard cuvette spectrophotometer Operation A 1 ul sample is pipetted onto the end of a fiber optic cable the receiving fiber A second fiber optic cable the source fiber is then brought into contact with the liquid sample causing the liquid to bridge the gap between the fiber optic ends The gap is controlled to both 1mm and 0 2 mm paths A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a
37. able surface tension in the samples to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning 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 measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Measurement Concentration Range The NanoDrop ND 1 000 Spectrophotometer will accurately measure protein samples up to 20 mg ml BSA without dilution A table of concentration range and typical reproducibility is listed below 9 1 Section 9 Proteins amp Labels l Approx Typical Reproducibility e H Upper minimum 5 replicates yp Limit SD mg ml CV Purified BSA 0 10 mg ml 20 mg ml sample range 0
38. ady for quantitation of protein samples at startup This module displays the UV spectrum measures the protein s absorbance at 280 nm A280 and calculates the concentration mg ml Like the Nucleic Acid module it automatically switches to the 0 2 mm pathlength at very high concentrations of protein Also analogous to the Nucleic Acid module the Protein A280 module displays and records 10 mm 1 cm equivalent data on the screen and in the archived data file Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension That occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning 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 measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface prop
39. alibration Check will be resetto the starting conditions and any previous measurements discarded Open Cleaning Procedure This warning occurs when a possible problem with the column is detected The software compares the long path and short path absorbances and issues a warning to the user if the short path is not 20 of the long path absorbance within a tolerance The most common explanation is that the column is not forming properly due to the pedestal being unconditioned When a pedestal becomes unconditioned sample droplets applied to the bottom pedestal will flatten out and cover the entire pedestal surface rather than Dead up Buffers containing detergents and various other reagents may cause the pedestal surfaces to become unconditioned We have noted that routine use of the Bradford reagent may result in difficulty forming columns with 1 ul samples Reconditioning Use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement 1 Open the vial containing PR 1 and use the applicator provided in the kit to remove a pin head sized amount of the compound Note Additional applicators may be purchased from VWR 2 Apply a very thin even layer of PR 1 to the surface of the upper and lower pedestals 3 Wait 30 seconds for the PR 1 to dry 4 Fold a clean dry laboratory wipe into quarters and remo
40. atb dolmirnbod Export Report amp imi filo of the displeyod Aeport amp Standes Standands Tables Tables surindle tor import no Erel 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 ndv 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 NanoDrop Data Reports folder and right click on the file of interest Additional features of the Report page Test Type Automatically populated with data method type e Date and time Auto fills in the date and time when a report is generated Report Name User defined designation for the current report Report Full Mode Drop down box defining options for managing reports The four options include Ignore Save Print Save and Print The selected action will be executed when the report reaches the designated Max Report Size Max Report Size Default number is set at 200 Note This feature is not utilized if the Report Full Mode is set to Ignore Standards Page The Standards page will display
41. ated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous nucleic acid samples containing incorporated fluorescent dyes However if you are unsure about the surface tension properties of your sample or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample Measurement Concentration Range The NanoDrop ND 1000 Spectrophotometer will accurately measure fluorescent dye and nucleic acid concentrations up to 100 pmols ul Cy3 and 750 ng ul DNA respectively without dilution A table of sample concentration ranges is listed below Sample Detection Limit Approx Upper Typical Reproduc ibility Type pmol ul Limit pmol ul minimum 5 replicates SD pmol ul CV Cy3 Cy3 5 Alexa Fluor 555 sample range 0 20 4 0 pmol ul sample range gt 4 0 pmol ul 2 Cy5 Cy5 5 and Alexa Fluor sample range 0 12 2 4 pmol ul 647 0 12 0 12 pmol ul sample range gt 2 4 pmol ul 2 Alexa Fluor 488 and Alexa sample range 0 40 8 0 pmol ul Fluor 594 0 40 pmol ul sample range gt 8 0 pmol ul 2 sample range 0 30 6 0 pmol ul Alexa Fluor 546 0 30 pmol ul sample range gt 6 0 pmol ul 2 6 1 Section 6 MicroArray Baseline Calculation amp Normalization The software normalizes the visual spectrum display for all readings at 750nm and will automatically calculate a baseline between 400 and 7
42. by clicking on the preferred option or by scrolling through the selections using the up or down arrow keys located to the left of the sample type box A description of each sample type is given below TAbs Tmgimk A general reference setting based on a 0 1 1 mg ml protein solution producing an Absorbance at 280 nm of 1 0 A where the pathlength is 10 mm or 1 cm il AGA Bovine Serum Albumin reference Unknown sample protein concentrations are calculated using the i mass extinction coefficient of 6 7 at 280 nm for a 1 10 mg ml BSA solution al IgG IgG reference Unknown sample protein concentrations are calculated using the mass extinction l coefficient of 13 7 at 280 nm for a 1 10 mg ml IgG solution Lysozyme e Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution User entered values for molar extinction coefficient M cm and molecular weight MW in kilo e PO 5000 Daltons for their respective protein reference Maximum value for e is 999 X 1000 and maximum MW kDa 50 00 value for M W is 9999 X 1000 j E User entered mass extinction coefficient L gm om h for a 10 mg ml 1 solution of the respective Pa coot E1 260 3 reference protein and Abs current value of the user selectable wavelength cursor and corresponding absorbance The wavelength can be set by dragging the cu
43. cedure for protein quantitation Like the BCA and Bradford Assays the Modified Lowry Assay requires standard curve generation each time it is run before unknown proteins can be measured The Modified Lowry procedure involves reaction of protein with cupric sulfate in alkaline solution resulting in formation of tetradentate copper protein complexes The Folin Ciocalteu Reagent is effectively reduced in proportion to the chelated copper complexes resulting in a water soluble blue product that is measured at 650 nm and normalized at 405 nm Pre formulated reagents utilized in the assay are available in kit form from numerous manufacturers Follow their recommendations when mixing the respective reagents at the time the assay is to be performed Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning 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 measurement
44. dards Terin gt Lory Eis Er gud Crea Ed Up to 5 replicates of each of up to 7 di bis id standards can be ue measured The software will not allow measurement of samples until a minimum of 1 standard and references or 2 standards are Mao de dla de io sds sia cis ole eds ein cle rin reg mami 2000 measured io ea Kee J Polynomial curve fitting requires more standard points Abtorboree G ESO rs 00 Step 3 Measure Samples em Ems Sinner Caen Help Sample SSC concentrations can be Sege Sanden calculated by using Sample D omy linear interpolation point to point between the two standards flanking the unknown sample or by using polynomial fitting Note In order Ahiahi Um to obtain a Abeocharce E res D DEZ concentration vaite aio di di d sop shs ar sis ein eds so dhe odo z O mami 1 503 oe ME a 1410007659150 alma iia sil unknown must fall within the limits of the standard curve Standard Curve Features Standard curves can be saved and reloaded for reference use by using the Standard Curve pull down menu and choosing the Save as or Load Standard Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time New to version 3 5 1 is the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete Standard Points
45. ders 2 The log files have been removed from the folder C NanoDrop Data log files Replace the log files if they have been moved Ifthe log files can not be located reinstall this software 3 The log files described above are setto read only Check the properties on each log file and ensure that the Read only box is unchecked This error code is most likely to occur if the Windows account that is currently logged into Windows does not have read and write access to the folder c nanodrop data or one of its subfolders See your PC administrator to make sure that all users of the NanoDrop software operate with a Windows account that has the appropriate access 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 Labview Runtime engine by selecting Start gt Programs NanoDrop gt Utilities gt Runtime Installer Report Format Loading Error This error occurs when the user does not have Write access to one of the report format files located at c nanodrop data custom report formats or the file has been moved from this folder Contact your PC administrator to give all users Read and Write access to this folder Replace the files if they have been removed If the file cannot be located reinstall the software Other Software Error Messages Can t find file OOIDRV INI This error occurs when trying to
46. e 220 750 nm Wavelength Accuracy 1 nm Wavelength Resolution 3 nm FWHM at Hg 546 nm Absorbance Precision 0 003 absorbance 1mm path Absorbance Accuracy 2 at 0 76 absorbance at 257 nm Absorbance Range 0 02 75 10 mm equivalent absorbance Detection Limit 2 ng microliter dsDNA Maximum Concentration 3700 ng microliter dsDNA Measurement Cycle Time 10 seconds Dimensions 14 cm X 20 cm Weight 1 6 kg Sample Pedestal Material of Construction 303 stainless steel and quartz fiber Operating Voltage 12 Vdc Operating Power Consumption 6 W Standby Power Consumption 1 5 W CE Approval Units sold in Europe Australia and New Zealand UL CSA Approval Units sold in N America S America Asia and Africa Included in system software compatible with Windows 2000 or XP Blanking and Absorbance Calculations When the NanoDrop ND 1000 Spectrophotometer is blanked a spectrum is taken of a reference material blank and stored in memory as an array of light intensities by wavelength When a measurement of a sample is taken the intensity of light that has transmitted through the sample is recorded The sample intensities along with the blank intensities are used to calculate the sample absorbance according to the following equation Absorbance log Intensitysampie Intensitypiank Thus the measured light intensity of both the sample and of the blank are required to calculate the absorbance at a given wavelength Concentration Calc
47. e NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer using the regular Bradford assay unknown protein concentrations from 100ug ml up to several thousand micrograms ml ug ml can be determined The best linearity is in the 100 1000 ug ml range A mini Bradford assay covers the approximate range of 15 125 ug ml Coomassie dye dye and Coomassie dye protein aggregates are frequently encountered in Coomassie dye based protein assays With time particulate can be observed which can cause significant fluctuations in Absorbance readings It is also important to note the total analyte protein dye signal at 595nm is limited to 0 150 A as a result of the 1 0mm pathlength of the instrument the Bradford Coomassie dye reagent concentration and the acidic pH Making measurements in triplicate of standards and samples unknowns is good practice particularly with the limited assay signal obtained with the Bradford Assay Assa Approx Approx Typical Reproducibility T e Lower Upper minimum 5 replicates om Limit Limit SD ug ml CV sample range 100 500 ug ml 25 ug ml Regular Bradford Bradford 100 ug ml 8000 ug ml sample range 500 8000 ug ml 5 sam
48. e a new baseline The absorbance value of the baseline is subtracted from the absorbance of the specirum Max Absorbance used to rescale the upper limit of the vertical axis Hi Abs samples with high absorbance up to 75 A equivalent 10 mm path can be measured directly applicable for instruments with serial numbers gt 500 only or that have been field retrofitted This capability is selected by choosing the Hi Abs button on the header bar When this is selected the absorbance is measured using the short path 0 2mm and plotted as a red line normalized to a 0 1mm path for easier visual comparison Sample ID label will be stored with sample data in the file folder C WanoDrop Data User name HiAbs This data cannot be imported into the Data Viewer but can be opened with Excel type spreadsheets This feature may be selected as the default option using the User Preferences module 7 1 Section 7 UV Vis Normalize This is a user selectable feature in this module If selected the software will automatically normalize the spectrum based on the lowest absorbance value in the range 400 750 nm This feature may be selected as the default option using the User Preferences module 1 2 Section 8 Protein A280 8 Protein A280 Proteins unlike nucleic acids can exhibit considerable diversity The A280 method is applicable to purified proteins exhibiting absorbance at 280nm It does not require generation of a standard curve and is re
49. e software does not start properly refer to the Troubleshooting 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 size 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 2 1 Section 2 Initial Set Up Software Upgrades NanoDrop Technologies makes periodic upgrades to the NanoDrop software These upgrades are available for download at www nanodrop com Cable Connections To make measurements with the instrument connect the USB cable to instrument and the PC plug in the 12V power supply and connect to the power input at the back of the instrument Note The power supply can remain plugged into the NanoDrop ND 1000 Spectrophotometer while the instrument is not in use When the unit is in this standby mode power consumption is 1 5 W and the flashlamp is not energized Also the instrument does not utilize a power switch or give a
50. e will guide you to measure your reference then at least one standard before allowing measurement of samples Reference samples refer to the dye reagent without any protein added ie the O sample Replicate counter for tracking replicate number during reference and standard measurement Reset all Standards F11 clears all replicates of all standards Reset 1 Standard F12 clears all replicates of the selected standard Absorbance at 595 nm the protein dye complex s absorbance at 595 nm for the 1mm pathlength Cursor A and Absorbance this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance The cursor wavelength can be set by the user Note The user selected wavelength and absorbance are not utilized in any calculations ug ml concentration of the sample unknown Making Bradford Protein Measurements A standard curve is required every time the Bradford assay is run Although curves can be saved and reloaded in the NanoDrop ND 1000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay Additionally a standard curve set up may be reloaded This feature will recall the respective standard series used in a previously saved standard curve Both single and multi point standard curve generation is incorporated into the software A standard curve can be developed using a reference Modified B
51. easurement 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 pedestal After measuring a large number of samples however it is recommended that the areas around the upper and lower pedestals be cleaned thoroughly 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 Note Please do not use a squirt bottle to apply de ionized water Decontamination of Measurement Pedestals If decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach solutions freshly prepared can be used to ensure that no biologically active material is 3 1 Section 3 General Operation 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 Note Please do not use a squirt bottle to apply diluted bleach Special Cleaning Requirements for Proteins Proteins and solutions containing surfactants can un condition the measurem
52. ent pedestal surfaces so that the liquid column does not form well with 1ul samples If this occurs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Sample Size Requirements Although sample size is not critical it is essential that the liquid column be formed so that the gap between the upper and lower measurement pedestals is bridged with sample Field experience indicates that the following volumes are sufficient to ensure reproducibility e Aqueous solutions of nucleic acids 1 ul e Purified protein 2 ul e Bradford BCA or Lowry assay 2 ul e Microbial cell suspensions 1 2 ul lt 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 1000 Spectrophoto
53. erties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Measurement Concentration Range The NanoDrop ND 1 000 Spectrophotometer will accurately measure protein samples up to 100 mg ml BSA without dilution To do this the instrument automatically detects the high concentration and utilizes the 0 2mm pathlength to calculate the absorbance A table of concentration range and typical reproducibility is listed below Typical Reproducibility Sample Type ers eee minimum 5 replicates SD mg ml CV Purified BSA 0 10 mg ml 100 mg ml sample range 0 10 10 mg ml 0 10 mg ml l sample range gt 10mg ml 2 Unique Screen Features Proteins A280 File Euil Fin Biana 1 07 PM User Detault Ceres contol Clear graph each Sample H nmnomalzation Loch 13 325 Sample ID ER E CH Eamplaz 2 Ek LP La n 2 A ei A E E ds a 7 095 228010 men ppib 0453 up chu ell O74 hice EE 060 270 280 290 300 310 320 330 340 350 ca d maim AS Wivelengih nm gim 1 d AS ERRIO 1 524 Sample Type There are six sample types options available for purified protein analysis and concentration measurement All of the options can be viewed by clicking the mouse while it is positioned within the Sample Type box 8 1 Section 8 Protein A280 The sample type color keyed can be selected
54. f the User s Manual available from Main Menu 16 2 This error occurs because no light or not enough light is reaching the detector If the troubleshooting steps outlined in the message do not fix the problem perform an Intensity Check Open the Utilities and Diagnostics module from the main menu With the sampling arm down select OK to initialize the spectrometer and then select Intensity Check You should see a red and black spectrum and a bias value greater than 65 as shown below This indicates that the USB communication is normal the power supply is operational and the flashlamp is functioning Diagnostics File Show Context Help Intensity Check DetectorBias 156 4000 0 3500 0 3000 0 11 Jl 2500 0 A VI se 1 L L JW Aa ll i 1500 0 JN At Jad Vt AA l DR W TN D 1000 0 a 500 0 4 0 0 220 0 300 0 350 0 400 0 450 0 500 0 550 0 600 0 650 0 700 0 750 0 Wavelength nm Serial Number USB2E3147 A152 Configuration 1 000000 1 1 7 48 24 If no spectra appear in the image confirm that the power supply is firmly connected to the instrument and the plug is connected to a working outlet Next confirm that the power supply is operating properly To do this connect the leads of a volt ohmmeter to the outlet of the supply The voltage should be 12 20 Vdc center positive If none of the troubleshooting steps above solve the problem refer to the Technical Service section for getting
55. f an undiluted sample measured on the ND 1000 is at the same pH as the diluted sample measured on the second spectrophotometer William W Wilfinger Karol Mackey and Piotr Chomczynski Effect of pH and lonic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity BioTechniques 22 474 481 March 1997 Wavelength accuracy of the spectrophotometers Although the absorbance of a nucleic acid at 260nm is generally on a plateau the absorbance curve at 280nm is quite steeply sloped A slight shift in wavelength accuracy will have a large effect on 260 280 ratios For example a 1 nm shift in wavelength accuracy will result in a 0 2 change in the 260 280 ratio Since many spectrophotometers claim a 1 nm accuracy specification it is possible to see as much as a 0 4 difference in the 260 280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are both within wavelength accuracy specification Nucleotide mix in your sample The five nucleotides that comprise DNA and RNA exhibit widely varying 260 280 ratios The following represent the 260 280 ratios estimated for each nucleotide if measured independently Guanine 1 15 Adenine 4 50 Cytosine 1 51 Uracil 4 00 Thymine 1 47 The resultant 260 280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260 280 ratios for the four nucleotides present It is important to note that the generally accepted ratios
56. facturer for the Modified Lowry method Follow the manufacturer s protocol for the assay including recommended incubation times and temperature Additionally use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the ND 1000 can measure higher protein concentrations you may need to supply your own protein standards at higher concentrations than provided by the manufacturer Unique Screen Features View Standard Curve F8 selecting this button allows the user to view the standard curve at any time Sample Type choices are Reference Standards 1 7 and Sample The software will guide you to measure your reference then at least one standard before allowing measurement of samples Reference samples refer to the dye reagent without any protein added ie the O sample 11 1 Replicate counter for tracking replicate number during reference and standard measurement Reset all Standards F11 clears all replicates of all standards Reset 1 Standard F12 clears all replicates of the selected standard Absorbance at 650 nm the Cu complex s absorbance at 650 nm for the 1mm pathlength Cursor A and Absorbance this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance The cursor wavelength can be set by the user Note The user selected wavelength and absorbance are not utilized in any calculations mg ml the concentration of the sample
57. g 2ul samples To print a copy of the results for your records click the Print Screen button A JPG of the final results is automatically archived on the hard drive at C NanoDrop Data Calib check If recalibration is required please contact NanoDrop Technologies Y 302 479 7707 or send at email to info nanodrop com 15 1 Section 15 Calibration Check NOTE The CF 1 Calibration Fluid is supplied in a single use vial The CF 1 must be used within one hour of opening the vial Exposure to the environment or transferring of the fluid to another container may cause a significant change in concentration 15 2 EE 16 Troubleshooting Error USB2000 Error USB2000 Unable To find Device with Serial This error might appear upon software startup and usually indicates that the USB cable is not properly connected or the software is not loaded properly To troubleshoot do the following 1 Confirm that the USB cable is connected to both the PC and the instrument 2 lf 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 from USB Reset After unplugging and then reconnecting the USB cable the Found New Hardware Wizard should start as shown below Windows XP SP2 o
58. ges of the sample retention system is that samples can be recovered from the upper and lower measurement pedestals by extraction with a pipette 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 1000 version 3 2 Section 3 General Operation E ND 1000 3 3 1 Liser Preferences Lies amp Ciegnosbes Application Modules The NanoDrop software has been tailored to meet the life scientist s needs It includes the following application modules e Nucleic Acid concentration and purity of nucleic acid e MicroArray dye incorporation concentration and purity of nucleic acid e UV Vis general UV Vis measurements e Cell Cultures absorbance light scattering measurement of suspended microbial cells e Protein A280 concentration and purity of purified protein e Proteins amp Labels concentration of dye labeled proteins conjugates and metalloproteins e Protein BCA protein concentration using the BCA assay e Protein Bradford protein concentration using the Bradford assay e Protein Lowry protein concentration using the Modified Lowry assay User Preferences Each user has the option to configure a number of settings in the various application modules Some key preference options available for each of the User Preference tabs are as follows e Archiving I
59. hown below Power Options Properties 2 x Power Schemes Alarms Power Meter Advanced Hibemate E Select the power scheme with the most appropriate settings for this computer Note that changing the settings below will modify the selected scheme Save As Delete m Settings for Home Office Desk power scheme When computer is I Plugged in Running on batteries Turn off monitor After 20 mins DI After 15 mins DI Turn off hard disks Never DI After 10 mins DI System standby Never DI After 30 mins DI System hibernates Never Se after 45 mins v 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 PC it may be necessary to replace the USB card on the original PC Signal Error Signal Error This is most likely caused by Sample surface is dirty make sure surfaces are clean Sample arm make sure sample arm is in down position No power to instrument check 12 power supply For more details refer to the Troubleshooting section o
60. including recommended incubation times and temperature Additionally use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the ND 1000 can measure higher protein concentrations you may need to supply your own protein standards at higher concentrations than provided by the manufacturer Unique Screen Features View Standard Curve F8 selecting this button allows the user to view the standard curve at any time 10 1 Section 10 Protein BCA Sample Type choices are Reference Standards 1 7 and Sample The software will guide you to measure your reference then at least one standard before allowing measurement of samples Reference samples refer to the dye reagent without any protein added ie the O sample Replicate counter for tracking replicate number during reference and standard measurement Reset all Standards F11 clears all replicates of all standards Reset 1 Standard F12 clears all replicates of the selected standard Absorbance at 562nm the Cu BCA complex s absorbance at 562 nm for the 1mm pathlength Cursor A and Absorbance this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance The cursor wavelength can be set by the user Note The user selected wavelength and absorbance are not utilized in any calculations mg ml the concentration of the sample unknown Making BCA Measurements A standard c
61. ins such as hemoglobin using wavelength ratios Fluorescent Dye Selection There are currently nine fluorescent dyes that are hard coded for use with the Proteins and Labels module see table below Users can also enter amp save fluorescent dyes not coded within the ND 1000 software using the Dye Chromophore Editor button found in the main menu Dyes can be selected using the scroll arrows or by highlighting the Dye 1 or Dye 2 box The respective absorbance wavelength extinction coefficient and 280nm corrections will be automatically utilized for measurement and concentration calculation The default settings from NanoDrop remain Dye 1 set to Cy3 In addition to the fluorescent dyes available from the drop down menu an option entitled None is also available Selecting None disables the respective calculations amp numeric displays corresponding to that dye Note Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes Dye Chromophore List Editor Dye Chromophore List 260 nm 280nm Insert Below Delete Alexa Fluor 546 Selected Alexa Fluor 555 4 Alexa Fluor 594 Edit Selected Alexa Fluor 64 Alexa Fluor 660 0 35 055 Note predefined dyes are indicated with a diamond and cannot be modified Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to vari
62. l peaks Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring most aqueous samples If you are unsure about your sample composition or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path completely is covered by sample Measurement Concentration Range All ND 1000 spectrophotometers will measure absorbance up to the 10mm pathlength equivalent of 15 A ND 1000s with serial numbers gt 500 or those that have been field retrofitted can utilize the short path length 0 2mm which enables the 10mm pathlength equivalent of 75 A Unique Screen Features T UN Vis Module File Eda Help Re blank Print Screen Recording Measuromornt completo 6 14 2007 1259 PM Measure Blank Print Repot Show Report sor Detauli a Normolize v HiAbs I7 0 gt Max Absorbance On On Sample iD FITI Sample Baseline 0 000 207 LU y U U U J 220 250 300 35 450 500 550 251 B 87545 0111 48 18 Wavelength nm 11 Abs1 and 12 Abs82 current values of the user selectable wavelength cursors and corresponding absorbencies for a 1 mm pathlength The wavelengths can be set by dragging the cursor using the up down arrows or typing in the desired wavelength Baseline the absorbance of the user selectable baseline horizontal cursor The user may drag this cursor to a new vertical position to creat
63. meter s measurement pedestals is easily addressed Simple wiping of the upper and lower measurement pedestal with a dry laboratory wipe is highly effective in eliminating carryover for samples differing in concentration by as much as three orders of magnitude see data at www nanodrop com This is possible since each measurement pedestal is in actuality a 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 spectrophotometry Genomic DNA lambda DNA and viscous solutions of highly concentrated nucleic acids 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 Effect of Evaporation and Solvents Evaporation of the sample during the measurement cycle usually has just a minimal effect on absorbance readings and may result in a 1 2 increase in sample concentration This can be observed in the field by measuring the same sample successively over time Highly volatile solvents such as hexane will likely evaporate before the measurement can be completed Less volatile solvents such as DMSO can be used successfully Sample Recovery One of the advanta
64. n addition to the primary data storage of archive files at c nanodrop data users may elect to save their data to an additional location This option can be chosen under the Archiving tab by selecting the Duplicate data storage box 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 and Exit button before exiting the User Preferences module The user may also elect to deselect an automatic prompt to close the Data Viewer whenever a module is closed The Data Viewer must be closed if a different module is opened before data can be reviewed e Reports Users may choose to select the Auto Reporting option for any of the application modules The auto reporting option allows data to automatically be saved to the report for all samples Users may choose this option under the Report tab by selecting the corresponding box next to the modules listed under Auto Reporting Save the auto reporting functions by clicking on the Save Preferences button before exiting the User Preferences window Note User preferences are stored in a log file When upgrading to a newer version of the software this file should be preserved If after upgrading to a new software version the user preferences do not appear correctly the log file should be manually copied to the proper directory See Passwords log for more detail e Nucleic
65. ncnnonnancnnnnnncnnonnnnos 10 1 BCA Kits Protocols and Sample Preparation cccccoocccnncccooncnnccnonons 10 1 Unique Screen Features A 10 1 Making BCA Meaesuremente 10 2 Standard Curve Features nnnnennnnnnnneennnnnnensnnnrnnrnnsennrrrrensennnrrreennne 10 3 Delete Standard Point sirere a a a ie 10 3 Exiting the BCA Module isnan e ane eara E EEA 10 4 Protein LOW VA a 11 1 Sample Volume Heouremente 11 1 Pedestal Hecondttonimg 11 1 Measurement Concentration Range oooooccccnncccncnonccnnnncnonnnannnnnnnnnnnnnnnaos 11 1 Modified Lowry Kits Protocols and Sample Preparati0N 11 1 Unique Screen Features ennio ea inaa i e ae ae a aeiia 11 1 Making Lowry Measurements ccccsecccccseeeeceeeeeeceeeeeeeeeeeesseeeeesaeeeeens 11 2 Standard Curve Features oooooncccnncccccconncnnnncnnnnnnnncnnnnnonnnnnnnnnnnnnnnonanenenns 11 3 Delete Standard POINTS nnrir a a a 11 3 Exiting the Lowry Module 000 dida 11 4 Protein Bradford ca aa 12 1 Sample Volume REQuireMentt cccccceeceeeeeeeceeeeeeeaeeeeeeeeessaeaeeeeeeees 12 1 Pedestal Hecondttonimg 12 1 Measurement Concentration Range oooooccccncccccnnoncccnnncnnnnnnnncnnonnnnnnnnnnos 12 1 Bradford Kits Protocols and Sample Preparation nnnen00nnnnn0nnnnnann 12 1 Unique Screen Features ccccccocccnccconcccnnnonnncnnnncnnonnnncnnnnnnnnnannnnnnnnnnnnananos 12 2 Making Bradford Protein Measurement cccoocccccccccnccnncnncnnnn
66. ngs 4 Nucleic Acid Folder 10 15 2004 2 08 PM E B drivers Microarray File Folder 10 15 2004 1 38 PM flexi Lowry File Folder 10 15 2004 1 21 PM 386 Proteins amp Labels File Folder 10 15 2004 12 53 PM E 3 NanoDrop Data Cell Culture File Folder 10 15 2004 11 20 AM E D Administrator OBCA Protein File Folder 10 15 2004 11 01 AM Je ault z Quy Vis H be File Folder 10 15 2004 10 50 AM BCA Protein Ouv vis File Folder 10 15 2004 10 50 AM Bradford Protein A 280 File Folder 10 15 2004 10 27 AM Cell Culture Bradford File Folder 9 23 2004 10 59 AM 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 14 1 Section 14 Archived Data and Data Viewer All data are 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 imp
67. nnnonanononnnnnnos 12 2 Standard Curve Features nnnn0nnnnnennnennnnnnenennnrnrrnnsennrrrrensennrrrreennne 12 3 Delete Standard POS inicias 12 3 Exiting the Bradford Module ootiosinnioicin Add edd 12 4 Cel e DE 13 1 Sample Size Requirements occcccncccccccnnccnnccccnnonnncnnnnnnononanonnnnnononnanannnnnns 13 1 Cell Suspension Concentrations ssoo0annnnnenennnnnnennsennnnnrnnsennnnnnennene 13 1 Sample Homogeneity ccccccccccccncccnnccconononccnnnncnnnnonnnnnnnnonononnnnnnnononnnonanenons 13 2 Decontamination of Measurement Pedestals nnn0nnnnnennnnnnnnnnnnennnnnn 13 2 Archived Data and Data Viewer ccccccssssseeesesseeeseeesneeeeseeennneesees 14 1 Archive Fle Cr e ici 14 1 Data Storage Hera Yassin a EAN 14 1 A O lessiaetanae 14 2 Archive Fil Ren EE 14 5 Opening Archived Data with Spreadsheet Programs oooooooccccnccccccccnnnno 14 6 CalibratiGn Cheese gege ii 15 1 Beleeg 15 1 THOUDIGSNOOUING ocrais aE a aaraa aias 16 1 ESF Fo 200 RE 16 1 CONNECTION EKFOM eene Eege 16 2 Sl E E 16 2 saturated Detecta a o E 16 3 Liguid Column Breakage EE 16 3 Other Software Error Meseages 16 5 Sample Accuracy and Reproducibility ooccooooccncconcnnccconoonnnnos 16 6 SEENEN 16 7 Unusual Spectr seinen e EE 16 7 TECHNICI SAME aa 16 8 Maintenance and WarraNtY oooooccconnccccnnnnccnnoncncnnnnrnnnnnnrnnnannrrnnanrrnnnnannnns 17 1 IS ul Te BEE 17 1 SC lee EE 17 1 Warani E 17 1 No AAA a
68. 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 is 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 absorbance 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 selected samples in a table format The user may modify column configurations for each method type and save multiple customized formats MO TOOD hiria Vimar wi Die Configusios Dais Papo Help hpo AUTIN 1671 AM Pio Bepon T ti pp Gem 620200 Zl A Eur En Deeg A ar E i Some key options useful for the Reports page are accessible through the Report tool bar drop down Choosing the Configure Report option brings up the following box Ropart comfipunation editor Saloct the report columns to display and the order that the columns ould appear Change the order by selecting and d
69. orbance at the 1 mm pathlength are not utilized in any calculations Dye 1 or 2 user selected dye Abs Norm normalized absorbance of selected Dye at the 1 mm pathlength pmol ul concentration based upon selected Dye s extinction coefficient See Concentration Calculation Beer s Law in the appendix for more details on this calculation ng ul concentration of nucleic acids in the sample calculated using the absorbance at 260 nm minus the absorbance at 340 nm i e normalized at 340 nm and the nucleic acid analysis constant See Concentration Calculation Beers Law in the appendix for more details on this calculation 260 280 ratio of sample absorbance at 260 and 280 nm The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm See 260 280 Ratio section of the Troubleshooting section for more details on factors that can affect this ratio 6 2 Section 7 UV Vis 7 UV VIS The UV VIS Absorbance module allows the NanoDrop ND 1000 Spectrophotometer to function as a conventional spectrophotometer Sample absorbance is displayed on the screen from 220 nm to 750 nm and cursors permit the measurement of individua
70. ort 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 It may also be accessed from the Main Menu page An ND 1000 Spectrophotometer does not need to be connected to the PC to use the Data Viewer module 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 Report page whether accessed through the Main Menu or Show Report Note Recording rather than Start Report must be selected in order to access the Data Viewer via Show Report Tool Bar Features common to all three pages include s File Options controlled by this tool bar function include File Page set up Print Window and Save Window 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 Reports This tool bar function allows the user to select columns of interest to be included in a re
71. ose measurable on a standard cuvette spectrophotometer This makes it possible to directly monitor concentrated cell suspensions Since the entire spectrum is displayed diluted samples exhibiting very low Absorbance at 600 nm can be monitored at lower wavelengths for example 280 nm 13 1 Section 13 Cell Cultures Sample Homogeneity The user must be sure to homogeneously suspend the cells when sampling for absorbance measurements and read the sample immediately to avoid significant cell settling Vigorous mixing may be required particularly when measuring concentrated samples Decontamination of Measurement Pedestals If decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach solutions 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 very resistant to most common laboratory solvents see Solvent Compatibility appendix Note Please do not use a squirt bottle to apply either a cleaning solution or water to the pedestal 13 2 Section 14 Archived Data and Data Viewer 14 Archived Data and Data Viewer Sample data from all application modules are automatically stored in archive files and can be opened by either the integrated Data Viewer software program or spreadsheet programs such as MS Excel
72. ow the buffer is handled Refer to section 14 Data Viewer for additional details All data is stored in the archive file at c WanoDrop Data and in a duplicate location if selected in User Preferences Note The system is configured to work with the Dymo Label Writer 400 printing on 30256 2 5 16 X 4 shipping labels but can print to any printer connected to the PC Show Report F7 The user can display the entries comprising the current Sample Report at any time by selecting the Show Report button This function will enable the Data Viewer software described in section 14 Parameters specific for the individual application modules are populated for each individual Sample ID 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 Sample The Sample indicator is activated when a sample report is being recorded It indicates the sample number of the last sample processed in the current report and increments with each successive measurement until the sample report is fully populated The sample buffer limit can be modified on the report page Exit This command closes all application modules
73. perating 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 hound How Tisrdesrp Wizard hound Now hardware Wizard Weloome to the Found Now Hardware Wizard winders el seach lot came and wpdabed soltarse Es habra ee yor Compute ve fe hidro eschen LD a oA Pe radica L piits Wieb ihe eatin pour p iatisar Piesd a pihy poire Tha wiad helpa you pt patane for Miralo LIST visa U pour hedeae c mo mih n rglalcddsnn LO Lar Wind tna Lo Weber Melaia lo sesch lo EH da E disk Seng a z it CO Yen ge me ony A i h gt Cer now and grey ime connect a dece Wal ed apa ma fa ed o d Gh ho not ar ag T prisa pha ica A AG Facial rata hor alist or ppscihc location Ahane Copa yea tee 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 installed 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
74. ple range 15 50 ug ml 4 ug ml Mini Bradford 15 ug ml 100 ug ml samplerange 50125 udini 259 Bradford Kits Protocols and Sample Preparation Commercial Bradford Protein kit manufacturers typically outline procedures for two different concentration ranges e A regular assay using a 50 1 reagent sample volume ratio To accurately prepare standards we suggest using a minimum sample volume of 4 ul in 200 ul of Bradford reagent larger sample volume is preferable e A mini assay using a 1 1 reagent sample volume ratio To prepare sufficient volume of these 1 1 mixtures we suggest using a minimum of 10 ul of sample and 10 ul of Bradford reagent in a PCR tube Using the same pipettor for both volumes will eliminate any pipette to pipette accuracy differences 124 Section 12 Protein Bradford In addition to the kit reagents protein standards e g BSA for generating a standard curve are provided by the manufacturer Follow the manufacturer s recommendation using standard BSA dilutions that cover the analytical ug ml range of interest Note Since the ND 1000 Spectrophotometer can measure higher protein concentrations you may need to supply your own protein standards at higher concentrations than provided by the manufacturer Unique Screen Features View Standard Curve F8 selecting this button allows the user to view the standard curve at any time Sample Type choices are Reference Standards 1 7 and Sample The softwar
75. port See section on Reports Page for more detail 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 deselected Import To import samples for viewing select Import Samples from the Data drop down of either the Plots or Report pages within the Data Viewer software This will bring up a new window with an Import Folder box and a Directory Tree as shown seen in the following image import folder Phenol ries Data 4 Hald dos Pp Corra Ce Grat kay or peerless dreciory E tect ete magie rock DF eng ADA EET Directory Tree Selected Samples Smpn LL Des es t l do Ceea on eal da tee ee termal Import amp Return Cancel Import Features include Import folder Used to select folder from where data are imported Folder selection must be at the level of user or higher may not select an application or method folder within a user in this activity box 14 2 Section 14 Archived Data and Data Viewer 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 indi
76. r RNA ssDNA 33 for single stranded DNA or Other for other nucleic acids The default is DNA 50 If Other is selected the user can select an analysis constant between15 150 When navigating amongst the three general sample types within the Nucleic Acids module the last constant value entered within the Constant sample type will be retained See the Concentration Calculation Beer s Law Appendix for more details on this calculation A and Abs the user selected wavelength and corresponding absorbance The wavelength can be selected by moving the cursor or using the up down arrows to the right of the wavelength box Note The user selected wavelength and absorbance are not utilized in any calculations A260 10 mm path absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm path Note This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length A280 10 mm path sample absorbance at 280 nm represented as if measured with a conventional 10 mm path Note This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length 9 1 Section 5 Nucleic Acids 260 280 ratio of sample absorbance at 260 and 280 nm The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as
77. radford reagent only no protein and a single replicate of one standard The multi point standard curve generator allows a maximum of five replicates for each of seven different standards There is no set order in which standards must be run A blank must be measured before the standard curve may be generated It is advisable to use water as the blank and use the dye reagent without any protein added as the 0 or reference sample There are three general procedural steps to unknown protein concentration measurements The requisite order including generating the standard curve is as follows Step 1 Measure the Reference Bradford reagent a zero Standard Note The software will guide the user with instructions in the large text box on the right side of the screen 12 2 Section 12 Protein Bradford Step 2 Measure Standards Up to 5 replicates of each of up to 7 standards can be Hasta up m 5 rap oiha nandai Live 2 ul ramplas The ubi miy EA yen j Ve measu red man gears Shank oi arr faces e cree mo penal iced A ate The software will not EE allow measurement Seel 1000 of samples until a Sane minimum of 1 Faset an Sssdarpcl Hatata 1 Sos standard and AA q XRO references or 2 Cursor mi SG ES PE standards are measured Abeobonce SSma O10 Polynomial Curve ugmi 2103 fitting requires more standard points Step 3 Measure Samples E 22 Sample
78. ragging a column header Sting to ihe desired postin Report columns ample ID Selecting OK will return the user to the reports page displaying only the columns of interest Report Features include Sort Allows users to sort data by column example by date or sample name and by either ascending or descending order 14 4 Section 14 Archived Data and Data Viewer Save Report Format Saves the current report format as an ndf file for retrieval and future use To designate a saved report format as the default format exit to the Main Menu choose Users Preferences and click Reports Use the Select Default Report Format to see the list of saved formats available for the specific method type Load 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 Configuration drop down on the tool bar Save Report and Load Report There are several options for this feature as seen in the following window Choose how to save the report Choose this option t he able to reload the Full Report pa into he Dana Mapo r Ste labor dato Ex t Raport Chob40 his Option do porn ab delimited por R po tmi io of thin dinpeasod Aeport Table Table Only suitable for impart into Excel J Chopig fis Option bo upon
79. reen 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 previously described Change Password This module enables each user having an authorized account ID to change their respective password Note The administrator using the Options or the Modify User entries in the Account Management module establishes whether individual user passwords will expire and if so after how many days User Account Setup Edit the current user parameters and click Continue to save or Cancel to exit without Saving Active D lt User ID joel i Full Name same Password Passwor repeat Security level 5 d 10 23 05 506 AM E 6 14 2007
80. rier liquid used with your samples onto the lower measurement pedestal and lower the sampling arm into the down position 2 Click on the Blank F3 button 3 When the measurement is complete wipe the blanking buffer from both pedestals using a laboratory wipe 4 1 Section 4 Common Module Functions 4 Analyze an aliquot of the blanking solution as though it were a sample This is done using the Measure button F1 The result should be a spectrum with a relatively flat baseline Wipe the blank from both measurement pedestal surfaces and repeat the process until the spectrum is flat See Blanking and Absorbance Calculations in the appendix for more information on blanking and absorbance calculations Re blank F2 The Re blanking option F2 establishes a new reference blank that is used for the absorbance calculations of subsequent samples However unlike the Blank F3 function the Re blank feature recalculates the absorbance spectrum for the most recent sample and displays this on the screen When the Re blank function is used the following message appears Blank Applied To displayed Spectrum 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 Note The system is configured to work with the Dymo Label Writer 400 printing on 30256 2 5 16 X 4 shipping labels but can print on any printer connected to
81. rsor using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations A280 10 mm Path 10 mm equivalent absorbance at 280 nm for the protein sample measured A260 280 ratio of sample absorbance at 260 and 280 nm Spectrum Normalization The baseline is automatically set to the absorbance value of the sample at 340 nm which should be very nearly zero absorbance All spectra are referenced off of this zero All data are now normalized and archived in the same format The user may elect to turn off the baseline normalization which may result in the spectra being offset from the baseline I Proteins A280 Eno Evia Flip EM5 2007 12 20PH User Default Cheney contol Accomula e until class r H nm normalization i E DU RI H SL Sample ID 30 00 _ Sample a O f fi 5 CG EP D LL E E mm A 10 00 228070 men pal 24417 nan eaZ al 10 00 I D 1 E 1 i I 1 I 1 I I 1 20 230 240 250 260 270 260 290 300 310 320 330 340 350 mg ml 38 60 Wowelengih nm 151 6 62545 0024016 Note If the spectra baseline offset is significant the calculated protein concentration may be higher than the true value Some samples may exhibit a greater baseline offset than the example above 8 2 Spectrum Overlay Control Section 8 Protein A280 The user can display more than one spectrum in the same display using thi
82. s wavelength maximum of 562 nm and normalized at 750 nm Pre formulated reagents of BCA and CuSO utilized in the assay are available in kit form from numerous manufacturers Follow their recommendations when mixing the respective reagents at the time the assay is to be performed Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning 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 measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Measurement Concentration Range
83. s feature The current sample plot will be displayed in bold and previous plots will be distinguished by different colors as seen in the following example Proteins A280 Eile Edit Help Gees Measurement complete 14 2007 1 18 PM Blank i FrintRepon i Show Renan User Detauh Laptop contol Accumulate until claar H Onmnomalization op 4 Serpe Type EE BSA Semple IL Nk Boo A V U 6 D n A S E E KN Al 280 abe 0966 228070 mm poh DAER ball to ahn 240 250 280 270 280 290 300 30 320 330 340 350 J I g m Wavelength nm g 351 0 0070 We CT KIT Eample 3 07 1 45 The default option is set to clear the display for the next reading The user may set the overlay control to clear after each sample plot default setting after each new report or accumulate plots until prompted to clear The Clear Now setting will clear all current and previous plots When the overlay function is active the software module will auto scale the y axis based on the sample with the highest absorbance at 280nm Note When the overlay function is active the Blank function doesn t clear the existing overlaid sample spectra 8 3 Section 9 Proteins amp Labels 9 Proteins amp Labels This software module can be used to determine protein concentration A280nm as well as fluorescent dye concentration protein array conjugates or to measure the purity of metalloprote
84. 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 Heat DNA samples to 55 C and vortex before measurement Due to the small volumes required by the ND 1000 it is extremely important to ensure that the sample being measured is homogeneous Field experience has shown that samples containing large molecules such as genomic or lambda DNA are particularly susceptible to this phenomenon Note Larger volumes used by cuvette based spectrophotometers will minimize or mask the effect of sample non homogeneity Perform a Blanking Cycle This will confirm that the instrument is working well and that any sample carryover from previous measurements Is not a concern To run a blanking cycle perform the following 1 Open the application software module 2 Load an aliquot of the blank the buffer solvent or carrier liquid used with your samples onto the lower measurement pedestal and lower the sampling arm into the down position 3 Click on the Blank F3 button to store the blank reference 4 Analyze a fresh replicate of the blanking solution as though it were a sample by selecting Measure F1 The result should be a spectrum that varies no more than 0 050 A 10mm absorbance equivalent 5 Wipe the blank from both measurement pedestal surfaces
85. t up under the File drop down menu Click on the Printer Set up button and then select the Dymo Printer under the Printer Name dropdown box Next click on Properties and select Advanced on the next window Select Paper size 30256 and click OK to save the selection Er 18 3
86. t your system administrator if this occurs Insufficient Memory This error message or others with similar wording occurs when attempting to install the operating software ona 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 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 Make sure the sample surfaces are clean before starting the software module A dirty sample pedestal on startup can cause erroneous absorbance readings 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 Note Do not use a squirt bottle to apply de ionized water Use a 1 5 2 ul sample 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
87. ta as an email attachment to your distributor or to info nanodrop com The archived file can be found at C WanoDrop Data gt User name gt Application Module BCA Protein Bradford Cell Culture Protein Lowry Proteins and Labels MicroArray Nucleic Acid Protein A 280 UV Vis or UV Vis HiAbs 16 8 EE WTA 17 Maintenance and Warranty Cleaning The primary maintenance requirement of the NanoDrop ND 1000 Spectrophotometer is keeping the measurement pedestal surfaces clean Upon completion of each sample measurement wipe the sample from the upper and lower pedestals to prevent sample carryover and avoid residue buildup It is also recommended that both measurement pedestals be cleaned with de ionized water upon completion of a testing series Apply 5 ul of dH20 solution onto each bottom pedestal Lower the upper pedestal arm to form a liquid column let it sit for approximately 2 3 minutes Wipe away the water form each upper and lower pedestal with a clean lab wipe Note Typically dH20 is sufficient for removal of samples that have dried on the optical pedestals There are a few cases i e dried proteins that may require a more rigorous cleaning protocol For these cases we recommend that 0 5M HCI be substituted for with 5 ul of dH20 to remove any residual HCI Please follow an HCI application with a water rinse to ensure any residual HCI is removed Note Do not use a squirt bottle to apply de ionized water or HCI Calibration
88. ted User Access Manager All gene Active Not Locket Mot Expired Never i Active Mot Locked Not Expired Never e Level 5 this is the security setting recommended for an ordinary user account An account with this access will be password protected and will be able to select specific user preferences Also all data generated will be automatically archived to the user s account in c Inanodrop data and the user specified location if that preference is selected 3 4 Section 3 General Operation Default level O security this access level is reserved for the Default account only This account enables any user without an account to access all the active software measurement modules Although it is not password protected user preferences can be set for this account All data generated will be automatically archived to the Default folder within the c Nanodrop Data folder Note For laboratories requiring that every user have a unique user account the administrator may disable the default user account Account Log in Log out and Time Out The user s account will remain active until 1 a user logs out of his her account by using the pull down menu to select either Default or another user name or 2 the user closes the software A user account may also be logged out automatically 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 sc
89. that are hard coded for use with the MicroArray module see table below Users can also enter amp save fluorescent dyes not coded within the ND 1000 software using the Dye Chromophore Editor button found in the main menu Dyes can be selected using the scroll arrows or by highlighting the Dye 1 or Dye 2 box The respective absorbance wavelength extinction coefficient and 260nm and 280nm corrections will be automatically utilized for measurement and concentration calculation The default settings from NanoDrop remain Dye 1 set to Cy3 and Dye 2 set to Cy5 In addition to the fluorescent dyes available from the drop down menu an option entitled None is also available Selecting None disables the respective calculations amp numeric displays corresponding to that dye Note Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes Dye Chromophore List Editor Dye Chromophore List Name Lem nm 260nm 280nm La ESE 0y3 1 50E 5 550 0 04 0 05 Below 4 Gab 2 50E 5 650 0 00 0 05 Alexa Fluor 488 710E 4 495 0 30 0 Delete Alexa Fluor 546 04E Selected Alexa Fluor 555 1 50E 5 55 D 0 0 Alexa Fluor 594 30E it lexa Fluor 647 Selected Alexa Fluor 660 e Cy3 5 4 Oy5 5 Note predefined dyes are indicated with a diamond and cannot be modified Sample Volume Requirements Field experience has indic
90. the NanoDrop ND 1000 Spectrophotometer path lengths of 1 0 mm and 0 2 mm are used compared to a standard spectrophotometer using a 10 0 mm path Thus the NanoDrop ND 1000 Spectrophotometer is capable of measuring samples that are 50 times more concentrated than can be measured in a standard spectrophotometer Note Absorbance data shown in archive files are represented as displayed on the software screen For Nucleic Acid Protein A280 and Proteins and Labels modules data are normalized to a 1 0 cm 10 0 mm path For MicroArray UV Vis Protein BCA Protein Bradford Protein Lowry and Cell Culture modules the data are normalized to a 0 1 cm 1 0 mm path For high absorbance UV Vis samples data are normalized to a 0 1mm path Solvent Compatibility The NanoDrop ND 1000 Spectrophotometer 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 HCl dilute HNO3 dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable Decontamination of Measurement amp Optical Surfaces If decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach solu
91. the actual reference standards applied to each particular sample at the time of measurement Note This page is available for software modules utilizing a Standard Curve fie Configuration Dita Reports Heb Pots Report Standards Test type Bradford Standards Rel Ayl Std 1 Sid 1 Sid 2 Sid 2 Sai 3 Sw 3 Sid 4 GA Get Alys r a r Archive File Converter All archive files located in the c Nanodrop Data folder generated with earlier versions of NanoDrop software will automatically be copied and converted to version compatible ndj files upon first use of the software Archive files generated with earlier versions of software and not stored in the folder ciNanoDrop Data will need to be converted 14 5 Section 14 Archived Data and Data Viewer manually before reviewing with the Data Viewer The File Converter can be accessed from the Import Data page and the conversion can be done one file at a time or by converting an entire file folder Note The original archive files are not altered during this process ND 1000 File Converter 3 5 1 This utility program converts ND 1000 archive files created with a previous version of the operating software to the current version 3 2 data format New files are created in the same folder as the source files The original files are left unchanged Select individual file to convert a Select folder to convert g Convert Folder Include all subfolders Opening Archived Data with
92. the instrument The number can be found on the bottom of the unit e JPG image of Utilities and Diagnostics module To get this open this module and select OK to initialize the module Select Intensity Check Once the spectrum has been created choose File gt Save Window as shown below Save to your hard drive and email as an attachment to your distributor or to info nanodrop com Diagnostics File Show Context Help Intensity Check Detector Bias 156 4000 0 3500 0 amo Al EU dg h A 2500 0 1000 0 Vi 1500 0 OU Hut LN Wu WA Lal 7 a K 500 0 4 0 0 220 0 300 0 350 0 400 0 450 0 500 0 550 0 600 0 650 0 700 0 750 0 Wavelength nm Serial Number USB2E3147 A152 Configuration 1 000000 1 1 7 48 24 e Application Module Screen Captures Screen captures of the actual spectrum as seen on your PC are of great use in diagnosing problems Making a screen capture is quite easy When in an application module press Alt Print Screen This copies the highlighted screen window to the PC s clipboard Next paste this screen capture into MS Word MS Paint this program usually comes standard with the PC and can usually be found in the Start gt Accessories menu or other graphics programs Save this as a jpg or doc file and send as email attachment to your distributor or to info nanodrop com Data Archive Files If you have questions about your data please send the archive file containing the suspect da
93. tions 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 Note Do not use a squirt bottle to apply the diluted bleach Setting Up a Dymo 400 Label Writer Printer To set DYMO default label to print with ND 1000 1 Open Control Panel then Printers and Faxes 2 Right click on Dymo printer then select Properties 3 Under General tab choose Printing Preferences then Advanced Then set the following e Paper Size 30256 e Halftoning Super Cell e Print Quality Barcodes and Graphics Click OK to close this window then click Apply Next click on the Advanced tab Ensure that Enable advanced printing features box is checked Click on Printing Defaults then Advanced and set the following e Paper Size 30256 e Halftoning Super Cell SPA 18 2 Section 18 Appendices e Print Quality Barcodes and Graphics 7 Click OK to close this window then click Apply 8 Click on the Device Settings tab and ensure 30256 Shipping Label is selected as the Default label It is best to confirm that the shipping label selection has been recognized by checking the set up from within the NanoDrop software Open the Data Viewer module and select Page Se
94. to be generated before unknown proteins can be measured The Bradford uses the protein induced absorbance shift of Coomassie Blue dye to 595 nm as a measure of protein concentration The bound protein dye complex is measured at 595 nm and normalized at 750 nm A single stabilized reagent mixture containing Coomassie Blue dye alcohol and surfactant in kit form is available from numerous manufacturers Follow the respective manufacturer s recommendations for all standards and samples unknowns ensuring they are subjected to the identical conditions and timing throughout the assay Sample Volume Requirement Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning 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 measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use th
95. tuzergent completa 6742007 1 13 Pa Fund Depot Show Fepon EAC Cite l Chumleu ont Clear graph each Sample Ve Sample Type Dime Sample ID dsDNA Sample A 210 mm Abs 17 55 GP TI men polh 2280 Wen path 200576 IS cela foe t i i i i D D i i i i i i 220 230 240 250 260 270 80 250 300 310 320 330 HI 350 Wavelength nri ngul 19509 151 0 RPO 0 eG Sample Volume Requirements Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous nucleic acid samples However if you are unsure about your sample or your pipettor accuracy a 1 5 2ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample Measurement Concentration Range The NanoDrop ND 1 000 Spectrophotometer will accurately measure dsDNA samples up to 3700 ng ul without dilution To do this the instrument automatically detects the high concentration and utilizes the 0 2mm pathlength to calculate the absorbance Detection Approx Typical Reproducibility Limit Upper Limit minimum 5 replicates ng ul ng ul SD ng ul CV 3700 ng ul dsDNA 2 3000 RNA sample range 2 100 ng ul 2 ng ul 2400 ssDNA sample range gt 100 ng ul 2 Unique Screen Features Sample Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 fo
96. ue Screen Features Cell Collores Module Pie blank Recording lvasiunramen complolo War 124 PH e Dora sampin IL Somple Baseline 0 000 Ft aen Aba Hl H O E 0 A A E E E User Cursor 450 500 3518 2871013401280 O NLL 600nm Absorbance current value of the absorbance at 600 nm with the Baseline absorbance subtracted Note the actual 1 mm absorbance is displayed and Abs current value of the user selectable wavelength cursor and corresponding absorbance The wavelength can be set by dragging the cursor using the up down arrows or typing in the desired wavelength Baseline the absorbance of the user selectable baseline horizontal cursor The user may drag this cursor to a new vertical position to create a new baseline The absorbance value of the baseline is subtracted from the absorbance of the spectrum Max Absorbance used to rescale the upper limit of the vertical axis Sample Size Requirements Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous samples However if you are unsure about your sample composition or your pipettor accuracy a 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path completely covered by sample Cell Suspension Concentrations Due to its shorter pathlength the ND 1000 can measure absorbencies that are 10 fold higher than th
97. ulation Beer s Law General The Beer Lambert equation is used to correlate the calculated absorbance with concentration A E b c Where A is the absorbance represented in absorbance units A E is the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of liter mol cm b is the path length in cm and c is the analyte concentration in moles liter or molarity M Fluorescent Dyes Microarray Measurement The NanoDrop software uses the general form of the Beer Lambert equation to calculate fluorescent dye concentrations in the Microarray module The table of extinction coefficients for each dye is below Extinction Measurement Dye Type Coefficient Wavelength liter mol cm nm Cy5 250000 650 Alexa Fluor 488 71000 Alexa Fluor 546 104000 Alexa Fluor 555 150000 18 1 Section 18 Appendices Alexa Fluor 660 132000 663 Nucleic Acids For nucleic acid quantification the Beer Lambert equation is modified to use an extinction coefficient with units of ng cm ml Using this extinction coefficient gives a manipulated equation c A e b Where c is the nucleic acid concentration in ng microliter A is the absorbance in AU e is the wavelength dependent extinction coefficient in ng cm microliter and b is the path length in cm The generally accepted extinction coefficients for nucleic acids are e Double stranded DNA 50 ng cm ul e Single stranded DNA 33 ng cm ul e RNA 40 ng cm ul For
98. urve is required every time the BCA assay is run Although curves can be saved and reloaded in the NanoDrop ND 1000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay Additionally a standard curve set up may be reloaded This feature will recall the respective standard series used in a previously saved standard curve Both single and multi point standard curve generation is incorporated into the software A standard curve can be developed using a reference BCA reagent only no protein and a single replicate of one standard The multi point standard curve generator allows a maximum of five replicates for each of seven different standards There is no set order in which standards must be run A blank must be measured before the standard curve may be generated It is advisable to use water as the blank and use the dye reagent without any protein added as the 0 or reference sample There are three general procedural steps to unknown protein concentration measurements The requisite order including generating the standard curve is as follows nua AA 0 TE Step 1 Measure the Reference BCA reagent a zero Standard Note The software will guide the user with instructions in the large text box on the right side of the screen vlt a a emm r E KAFI r ENTEN TE 450 400 500 5 50 560 5 600 60 140 560 6060 300 720 rey 19 10 E
99. ve the PR 1 by aggressively rubbing the surface of the upper and lower pedestals until all compound residue is removed Note The black appearance of the removed residue is normal The reconditioning process is complete once the laboratory wipe shows no more black residue To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up Additional information about the PR 1 kit may be found at www nanodrop com As an alternative to using the PR 1 Kit the pedestals may be reconditioned as follows 1 Fold a clean dry lab wipe over several times to increase its thickness 2 Press the lab wipe firmly down on the lower pedestal and buff rub very aggressively at least 50 times the lab wipe will rip during this procedure and will have to be refolded several times throughout the procedure The upper pedestal may also be buffed but care should be taken not to put too much force on the upper arm lf the warning persists and the user visually confirms that the liquid column is forming contact NanoDrop Technologies or your local distributor 16 4 Error Code 8 Error Code 8 Error reading or writing to file This might be caused by 1 The current Windows account does not have Read and Write priveleges to the folder CANanoDrop Datat and all of its subfolders Contact your PC administrator to give all users Read and Write access to these fol
100. vian ee ee ageet ees 4 3 Show Context Help CAHEN 4 3 Users Oe LE EEN 4 3 lte lg FE 5 1 Sample Volume Heouremente nanaisin a 5 1 Measurement Concentration Range ooooccccnncccccnnoccconnncnonnnancnnnnnnonnnannnnnns 5 1 Spectrum N rmalzalo cia at ta ita 5 2 Spectrum Overlay Control 5 2 MICPOA a EE 6 1 Fluorescent Dye Selection rcce an a a n R aa aeaa 6 1 Sample Volume Heouremente 6 1 Measurement Concentration Range cccsseeeeeeceeeeeeeeeeeeeeeeeseaeeeeeeeess 6 1 Baseline Calculation amp Normaltzaton 6 2 EE 7 1 Sample Volume Heouremente 7 1 Measurement Concentration Range ooooncccnnccccnnnoccccnnncnnnnnnnncnnnnnnnnnannnnnns 7 1 Unique Screen Features scsi dida 7 1 Protein A280 cnir 8 1 Sample Volume Heouremente 8 1 Pedestal ee tee Le EE 8 1 Measurement Concentration Range oooooccccnccccnnnoccccnnncnonnnannnnnnnnnnnnannnnnos 8 1 Unique Screen Features ias 8 1 Spectrum NormalliZa e EE 8 2 Spectrum Overlay Control 8 3 Proteins amp Babel Sin icc stessotc actin actin eee eee 9 1 Fluorescent Dye Selection enoe E 9 1 Sample Volume Heouremente 9 1 Pedestal Hecondttonimg 9 1 10 11 12 13 14 15 16 17 18 Measurement Concentration Range oooocccnncccccnonnccnnncnnonnnancnnnnononnnaanennns 9 1 Unique Screen Feature ide 9 2 ee Eer TE ee 9 2 Protein BCAA 10 1 Sample Volume Heouremente 10 1 Pedestal Hecondttonimg 10 1 Measurement Concentration Range oooocccnncccnccoonccnnn
101. vidual samples within a file or the entire file All import selections must be of the same application or method type Note Hi Abs data cannot be imported into the Data Viewer but can be opened with Excel type spreadsheets Archive File Converter ND 1000 data generated with earlier versions of NanoDrop software and not stored in c nanodrop data will need to be copied and converted to 3 2 version compatible ndj files Refer to the Archive File Converter section below for more details Note The original archive files will not be altered during this process 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 Sample Information and Spectrum Populated with the information associated with the most recently highlighted sample Import and Return Uses selected sample data to populate Plots and Reports windows Note Holding down the shift or control PC function keys will allow the user to select multiple samples and or files for importing The keys can also be used to deselect multiple samples as seen in the following example Import folder Spectrum EB Sample Information CipdancDeso hata Le 4 Haid dzen the Control of Sh ya eile talata AH eet n e dercor beg do tabi gp mubp e vebzschal ce a 10058 OF tameii cop
102. with a laboratory wipe and repeat the process until the spectrum is within 0 005 A 1mm path Confirm that reference blank solution and solvent are the same material Buffers often absorb in the UV range and therefore it is critical to blank the instrument with exactly the same material that the sample is suspended in Confirm that your sample is not too dilute 16 6 Measuring samples at or near the detection limit will result in measurements that can vary a significant amount Refer to the Measurement Concentration Range of the application module that you are using for the applicable measurement range Confirm instrument accuracy and reproducibility with CF 1 This is a potassium dichromate calibration standard available from NanoDrop Technologies and its distributors Itis a good practice to check the instrument s performance every six months with a fresh vial of CF 1 260 280 Ratio Many researchers encounter a consistent 260 280 ratio change when switching from a standard cuvette spectrophotometer to the NanoDrop ND 1000 spectrophotometer The three main causes for this are listed below Change in sample acidity Small changes in solution pH will cause the 260 280 to vary Acidic solutions will under represent the 260 280 ratio by 0 2 0 3 while a basic solution will over represent the ratio by 0 2 0 3 If comparing the NanoDrop ND 1000 Spectrophotometer to other spectrophotometers it is important to ensure that the pH o

NanoDrop ND-1000 Spectrophotometer manual

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