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NanoDrop 1000 Spectrophotometer V3.8 User`s Manual

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1. 800 1000 1200 1400 ug ml 13 2 Section 14 Cell Cultures 14 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 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 Unique Screen Features Cell Cultures Module File Edit Help Measurement complete 6 14 2007 1 24 PM s Exit User Default Sample ID Sample 1 Baseline 0 000 amp 00nmAbs 9 162 User Cursor A 280 lt inm Abs 1 832 250 300 350 400 450 500 550 600 650 700 Wavelength I 3 5 1 8 BB710 1 39 112 32 GE E 600nm Absorbance current value of the absorbance at 600 nm with the Baseline absorbance subtrac
2. it is the re analysis of the previous measurement with a new blank Plots Report Standards Test type Lowry 6 15 2007 11 00 AM Report Name Max Buffer Size 200 2 Buffer Mode Save Report amp Clear v Sample Date 1 Time 1 ng ul A260 3 A280 260 280 260 230 Constant 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 fl C NanoDrop Data Default lol x File Edit view Favorites Tools Help a Back gt i D P Search Ke Folders 3 x ie AA Address Ei C NanoDrop DatalDefault Go Norton AntiVirus E M Folders x Name 35 Size Type 5 Date Modfied amp C3 Documents and Settings af O Nucleic Acid File Folder 10 15 2004 2 08 PM a C3 drivers Microarray File Folder 10 15 2004 1 38 PM Q3 flexi Qlowry File Folder 10 15 2004 1 21 PM amp C5 i386 Proteins amp Labels File Folder 10 15 2004 12 53 PM D C3 NanoDrop Data Cel Culture File Folder 10 15 2004 11 20 AM E C3 Administrator BCA Protein File Folder 10 15 2004 11 01 AM zc mm CQUv vis Hiabs
3. NanoDrop 1000 Spectrophotometer V3 8 User s Manual The information in this publication is provided for reference only All information contained in this publication is believed to be correct and complete Thermo Fisher Scientific shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing performance or use of this material All product specifications as well as the information contained in this publication are subject to change without notice This publication may contain or reference information and products protected by copyrights or patents and does not convey any license under our patent rights nor the rights of others We do not assume any liability arising out of any infringements of patents or other rights of third parties We make no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Customers are ultimately responsible for validation of their systems 2010 Thermo Fisher Scientific Inc All rights reserved No part of this publication may be stored in a retrieval system transmitted or reproduced in any way including but not limited to photocopy photograph magnetic or other record without our prior written permission For Technical Support please contact Thermo Fisher Scientific 3411 Silverside Road Bancroft Building Suite 100 Wilmington DE
4. 32 bit only Windows 7 Professional 32 and 64 bit e 233 MHz or higher processor e CD ROM drive e 1GB of RAM for 32 bit and 2 GB for 64 bit system e 40 MB of free hard disk space e Available USB port the instrument can only be connected via the USB port 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 the operating 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 New Hartaare Wizard aeit welll aah on eua Targo dmit case wth o r Intro Page Windows XP SP2 All Windows Operating Systems Your NanoDrop 1000 Spectrophotometer should now be ready for operation If the software does not start properly refer to the Troubleshooting Section for pos
5. Acer Not Locked ot Expired epuer Not Locked ot Expired Never Not Lecke Not E VV 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 Nanodrop data and the user specified location if that preference is selected e 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 screen will appear indicating that the time is about to expire with a
6. BCA Kits Protocols and Sample Preparation 10 1 Unique Screen Features 10 1 Making BCA Measurements nn0nnnnnnannnnnnnnnnnnsnnnnnnsnnnrenrnrnnnnnnnnresennnen 10 2 Standard Curve Features sisi aa a a Ea 10 2 Delete Standard Pomte rieien iape ae a aa ET 10 2 Exiting the BCA Oe e EU 10 3 Protein Reg EE 11 1 Sample Volume Requirements nnne 11 1 Pedestal HeconditlonilQg eessen ee 11 1 Measurement Concentration Range ssssessssrrreereesrrrrrrreesrrrrrrrene 11 1 Modified Lowry Kits Protocols and Sample Preparation 11 1 Unique Screen Features 11 1 Making Lowry Measurements 11 2 12 13 14 15 16 17 18 19 Standard Curve EE 11 2 Delete Staridard Poltits noii Leto a ted 11 2 Exiting the Lowry Module nnnnannnnnnennnnnnnnnnnnsnnnnnnsnnnnnnsnnnrenrnrnnesenrrrennnne 11 3 Protein e e et 12 1 Sample Volume Heourement nnne 12 1 Pedestal Reconditioning cccccccseeeeeeeceeeeeeeesaeeeceeseeeeeeeeesaaeeeeeeaaaees 12 1 Measurement Concentration Hange sse 12 1 Bradford Kits Protocols and Sample Preparation 12 1 Ungues Screen Fealut6s uoi outer a eed as veda UE Dd eeuen s 12 1 Making Bradford Protein Measurements nnnnennnnnennenennennnnennnernrrrenene 12 2 Standard Curve Features nnns 12 2 Delete Standard PONS risane erine a a a 12 2 Exiting the Bradford
7. Measurement Type Sample Standards Step 3 Measure Samples Sample ID 030 Max Absorbance Sample concentrations can be calculated by using linear interpolation point to point between the two standards flanking the unknown sample or by using polynomial fitting Note In order to obtain a concentration value mg ml the sample unknown el es must fall within the limits of the standard curve Absorbance 0 014 1 mm Absorbance Absorbance 9595 nm 9 104 1 E H L Uu 600 625 650 675 700 725 1174 Wavelength nm ug ml 351 8 82545 0 02 48 16 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 Software versions 3 5 1 and higher include 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 12 2 Section 12 11BProtein Bradford standard all replicates of that standard are cleared by selecting Delete 1 Standard Additionally all standards can be deleted at on
8. the error persists contact your local distributor or Technical Support Liquid Column Breakage Warning The ratio between the long path and short path absorbance is out of tolerance This might be caused by 1 An air bubble was entrained in the sample Try measuring again 2 The liquid column is not forming can be confirmed visually If this is occurring try cleaning both measurement pedestals with water and then wiping each surface 50 times aggressively with a dry lab wipe If column breakage persisits use a larger sample size 1 5 2 ul 3 One or both of the measurement paths is out of calibration If this warning persists and the above remedies do not solve the problem contact NanoDrop Technologies or your distributor 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 2096 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 bead 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 resul
9. to keep our user list updated so that we may alert you to these updates and all information supplied is completely confidential You can register your instrument on our website 2 1 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 1 With the sampling arm open pipette the sample onto the lower measurement pedestal 2 Close the sampling arm and initiate a spectral measurement Le using the operating software on the measurement PC The sample column is di automatically drawn between the upper and lower measurement pedestals and the spectral measurement made lower measurement pedestal A 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 our website 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 measurement is usually sufficient to prevent sample carryover and avoid residue buildup Although generally not necessary 2 ul water aliquots can be used to clean the measurement surfaces after particularly high concentration samples to e
10. 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 3 4 Section 3 General Operation User Account Setup Edit the current user parameters and click Continue to save or Cancel to exit without Saving Active UserID joel i Full Name same Password Password 1 repeat Security level 5 Emir 1 10 23 05 506 AM 6 14 2007 CONTINUE CANCEL Passw
11. 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 description of Data Viewer below Mame Size Type Date Modified EI Nucleic Acid 2007 O06 13 v3 5 ndj 4KB NDI File 5 13 2007 7 53 AM sl Nucleic Acid 2007 06 O4 3 5 ndj 4KB NDI File 5 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 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
12. 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 up to 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 ja Step 1 Measure the Reference BCA reagent a zero Standard e Measurement Type C NN Note The software will guide the user with instructions in the LS Eo large text box on the right side of the screen Standard 1 Reset All Standards Delete 1 Standard Cursor m s 1 mm Absorbance Absorbance 0 000 Absorbance 562nm 0 000 0 05 450 480 500 520 540 560 580 600 620 640 660 580 700 720 750 mg ml 0 000 Wavelength nm gi 35 1 B 85342 0 43 96 24 6 14 2007 4 09 PM Detault Measurement Type Sample
13. 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 Software versions 3 5 1 and higher include 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 10 2 Exiting the BCA Module BCA Protein Std Curve Elle Show Context Help Reference mg ml Ave Abs 0 000 0 008 Abs 1 0 009 Abs 2 0 008 Abs 3 0 008 Standard 1 Standard 2 Standard 3 0 100 0200 0 500 0 003 0 012 0 039 0 004 0013 0 038 0 003 0014 0 040 0 002 0011 0 040 Standard 4 Standard 5 Standard 6 1 000 2000 4 000 0 081 0 152 0 279 0 081 0150 0 278 0 080 014 0 278 0 082 0 153 0 282 8 000 0 500 0 497 0 501 0 505 R squered 0 9998 User Default 6 5 2007 9 29 AM 351 4 B2545 0 11 64 24 St
14. D QU UU 18 1 CI AG EE 18 1 Waranty socal eee get hee ge cate T NE eee atte 18 1 Lee le e 19 1 Instrument Specifications 0 0 eee eeccccceeceeeeeeeeeeeeeeeeeeeeeeeeesesaaeseeeeeeeeesaas 19 1 Blanking and Absorbance Calculations esses 19 1 Concentration Calculation Beers Lawi 19 1 SOIVENT Eegiel TEE 19 2 Decontamination of Measurement amp Optical Surfaces n0000nnn00an 19 2 Setting Up a Dymo 400 Label Writer brmter 19 2 Section 1 OBOverview 1 Overview Instrument Description The Thermo Scientific NanoDrop 1000 Spectrophotometer measures 1 ul samples with high accuracy and reproducibility The full soectrum 220nm 750nm spectrophotometer 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 NanoDrop 1000 Spectrophotometer 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 pul
15. Measurement Exit User Default Measure F1 Each time a software module is opened initiated the Measure button is inactive as noted by its grayed out appearance A 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 Eile Edit Help Re blank A Print Screen Recording Make new BLANK 6 15 2007 9 36 AM Print Report Show Report EES User Default Overlay control Clear graph each Sample Sample Type RNA 40 Sample ID Sample 0 2 230 nm Abs 0 000 A 260 10 mm path NaN A 28010mmpath NaN 260 280 NaN 260 230 NaN 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm ng uL NaN 3 5 1 B B5342 0 43 96 24 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
16. Module 12 3 Protein Pierce 660 MM innn a S 13 1 Billie CR RE 13 1 Making Pierce 660 nm Protein Measurements nnn n 13 1 Standard Curve EMT 13 2 Delete Standard Poms EE 13 2 Gell e E 14 1 Sample Size Requirements neee A AERA 14 1 Cell Suspension Concentraions 14 1 Sample Homogeneity eeeeeessssssssesssseeeeeenennnn nennen nnn nnns 14 1 Decontamination of Measurement Pedestals ssuussss 14 1 Archived Data and Data VieWeD cscccseeesseeescessseesseeesseeseeeenseesees 15 1 Acne File Cre ALON EE 15 1 Data Storage Hierarchy sacerdo tti dein A eeu ase 15 1 CEET 15 1 Archive Ele CONV EE 15 4 Opening Archived Data with Spreadsheet Programs ssseeeeeeeeeeeenen 15 5 Galibfatlon CheGK dec 16 1 ed O10 10 6 Nite MEC PEE 16 1 TROUDICS NO OWING EE 17 1 Error USB 400 tH 17 1 COMME CIO qi iugo oasen denna ear Eege 17 1 SIOnal ttt necs stein np EE ELA D MES EM 17 2 Saluraled MDI I Ioro dm 17 2 Liquid Column Breakage ccccccececeseeeeeceeeecseeeeeceeeeesseeeeeeeeeeeeeseaeaeees 17 3 Other Software Error Messages nnnnenon0nnnnnnnnnennnnnnnnnrnnnrnnennennnnnnnee 17 4 Sample Accuracy and Reproducibility sseeeeeeeesssssse 17 4 260 290 RANG EE 17 5 Unusual Specl Un EE 17 6 Technical SUBD ON e M 17 6 Maintenance and Warranty eeeeeeeee eene eee nene nnn 18 1 CIC AN Le EN EE
17. Standards Reference and Standards EE Step 2 Measure Standards Reterence 0 000 5 0 008 Stas rd 1 0 100 5 Standard 2 0 200 Sende 088 5 003 Up to 5 replicates of each of up to 7 standards can be measured Standard 4 1 000 Standard 5 2 000 Standard 8 000 5 0 500 SS The software will not allow measurement of samples until a Sesasese seusesI minimum of 1 standard and references or 2 standards are Cursor Us s measured Polynomial curve fitting requires more standard points Absorbance 8 5 2 S x E E Absorbance 9 562nm 0 285 0 05 450 480 500 520 540 560 580 600 620 640 660 680 700 720 3 750 mg ml 4 000 sample spectrum 35 18 82545 0 03 48 24 evelength nm Ele Edit Standard Curves Help Re blank f PrintScreen Recording Stendera Cure Measurement Type Sample Standards Semple D m Step 3 Measure Samples Sample concentrations can be calculated by using linear interpolation point to point between the two standards flanking the unknown sample or 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 1 mm Absorbance Absorbance 9 562nm 9 337 0 05 450 480 500 520 540 560 580 600 620 640 660 680 700 720 780 mg ml 1 121 3518 82545 011 48 16 Wavelength nm Standard Curve Features Standard curves can be saved and reloaded for reference use by using the Standard
18. 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 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 contact your local distributor or Technical Support for assistance Technical Support If after referring to the above troubleshooting tips you are unable to resolve your problem please contact your local distributor or Technical Support for assistance The following information will be very helpful e Serial Number of 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 Save Window as shown below Save to your hard drive and email as an attachment to your local distributor or Technical Support Senal Humber WISE Pp E3545 Configuration EOFT e Dotechor Bes 273 Wavelengih Arrumecy Mme Single Ofiset 44 Manon Peaks Monitored 4840nm 4839 25 7nm ena SeSnm 15253 is i i i
19. 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 Section 15 Archived Data and Data Viewer ve Fie Converter Ls wv mee LO uon d Aen Flo Corworted Selected Samples Sempre O Oms 4 Tm la 11 1 2008 8155 AM 11 1 2008 OC AM AM k e T e 12 748 n3 s DNA e A7 pen OPa 11 3 2005 P Ai AM L e 4 ACR Search l e 19 7400 ng ul CINA 1M ONA Held dows Contol or Sha beya t select multiple rar Search is restected 10 fhe 191 Y cmenty salected tranch ofthe Import amp Retum l Cancel l 2 deecor bee Plots The Plots page displays selected sample spectra Text type Nucleic Acid Selected plot dimanea Poteet g ampla Infcemsaitorni do A agia Plot Features include Test Type Auto fills in module name Dat
20. first use of the software Archive files generated with earlier versions of software and not stored in the folder CANanoDrop Data will need to be converted 15 4 Section 15 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 gt Select folder to convert 3 Convert Folder Include all subfolders Opening Archived Data with 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 15 5 Section 16 Calibration Check 16 Calibration Check The Calibration Check is found within the Utilities and Diagnostics module and is accessed through the Main Menu It is used to
21. procedure The upper pedestal may also be buffed but care should be taken not to put too much force on the upper arm If the warning persists and the user visually confirms that the liquid column is forming contact your local distributor or Technical Support 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 C NanoDrop Data and all of its subfolders Contact your PC administrator to give all users Read and Write access to these folders 2 The log files have been removed from the folder C NanoDrop DateMog 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 17 3 Section 17 Troubleshooting 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 operating software have the appropriate Windows access level 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 engin
22. rise to variable surface tension properties 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 Solutions and reagents containing surfactants may 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 on our website Measurement Concentration Range When using a 20 1 reagent to sample volume dilution the BCA assay concentration range of detection is 0 20 mg ml to 8 0 mg ml on the NanoDrop 1000 Using a 1 1 reagent to sample volume dilution the concentration range of detection is 0 01 mg ml 0 20 mg ml Approx Approx Typical Reproducibility Assay Lower Upper minimum 5 replicates Type Limit Limit SD mg ml
23. spray the propellant into the instrument Procedure 1 Ensure the measurement pedestals are clean and that a 1ul water sample beads up on the lower pedestal 2 Remove any lint build up from around the instrument solenoid See the instructions at the end of this section for removal of lint build up 3 Open the NanoDrop 1000 Spectrophotometer Calibration Check Software and follow the prompts in the Customer Guidance text box of the software 4 Enter the Target Absorbance found on the CF 1 vial as directed in the image below Typically the target absorbance is 0 734 the actual value will depend on the lot of CF 1 Customer Guidance Measure M Amm E ENS ETT 7 e ege hd y TNZE e E 7 detects as Enter the target absorbance for CF 1 at 350 nm and 1 0 mm path Then add a 1uL s a ada water sample and select Blank Reset Instructions Measurementnm 350 Replicates 0 Corectionnm 600 1 0 mm Path 0 2 mm Path 0 000 0 000 NaN NaN NaN NaN NaN NaN 1 mm Path O2mmPath Measured Absorbance vs Target 70 10 1 1 1 1 D D 1 220250 300 350 400 450 500 550 600 650 700 750 Wavelength nm Oey 2 eo ET e 7 GE 3 3A152 1 17 48 24 Measurement 5 Add ul of deionized water and select Blank Section 16 Calibration Check 6 Before opening the ampoule of CF 1 Calibration Fluid shake vigorously to ensure solution is thoroughly mixed Ensure all sol
24. the problem contact Technical Support or your local NanoDrop products distributor Connection Error Connection Error There was an error communicating with the instrument Try the following Check the USB cable and select Retry It Retry fails disconnectthe USB cable and then reconnect and select Retry again If this does not salve the problem referto the Troubleshooting section ofthe 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 2 Control Panel Power Options The System Standby and System Hibernate should be set to Never for the Plugged In column as shown below Power Options Properties KAES Power Schemes Alarms Power Meter Advanced Hibernate wu Select the power scheme with the most appropriate settings for this computer Note that changing the settings below will modify th
25. 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 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 Dan Reports Hel Pots Report Standards Test type Bradlord Standards Archive File Converter All archive files located in the c Nanodrop Data folder generated with earlier versions of the operating software will automatically be copied and converted to version compatible ndj files upon
26. 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 Particulates often form with increasing development time which can cause significant fluctuations in Absorbance readings It is also important to note the total analyte protein dye signal at 595 nm 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 Ada Approx Approx Typical Reproducibility T 2 Lower Upper minimum 5 replicates yp Limit Limit SD ug ml CV sample range 100 500 ug ml 25 ug ml Regular Bradford 100 ug ml 8000 ug ml sample range 500 8000 ug ml 5 SCH sample range 15 50 ug ml 4 ug ml Mini Bradford 15 ug ml 100 ug ml sample range 50 125 ug ml 5 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 reagen
27. 19810 U S A Telephone 302 479 7707 Fax 302 792 7155 E mail info nanodrop com www nanodrop com For International Support please contact your local distributor Microsoft Windows Windows NT and Excel are either trademarks or registered trademarks of Microsoft Corporation in the United States and or other countries Adobe and Acrobat are trademarks of Adobe Systems Incorporated All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries NanoDrop is a trademark of Thermo Fisher Scientific Revised 12 10 Table of Contents 10 11 SUL ee 1 1 Instrument DesclpIOl c eccenc ceo eur Sina est loce tetbaruece ducati oram t eU ca labatur Ed 1 1 eeu Mc e TP Rer 1 1 PP PIICAIONS PED t 1 1 mci cmm TT 1 1 Dig e erc 2 1 Computer Requirements scere EEE A aaa i aa 2 1 Software installati n EE 2 1 Software Upgrades EE 2 1 Registering Your Instrument 2 1 e n rcenrirup m 3 1 The Sample Retention System cccccccsssseeccceeeesseeeeeeeeeeeseeeeeeeeeeeeeeaas 3 1 Cleaning the Sample Retention System EE 3 1 Software Architecture and Features snnneannnnnnnnennnnnnennennnrnnrenennnrnneee 3 2 User PrElEV ENG cc ens 3 3 Utilities and ACO e 3 3 Account Management 3 4 Dye Ghromopnore le 3 5 Common Module Functions ccccccscsesesseeeeeeseceesesseeeeeeeeeessneeeeseeees 4 1 Vill Lt le
28. 2 20 PM Measure Exit Print Repot 1 Show Report User Default Overlay control Accumulate until clear v 340 nm normalization C Off SampleType 85 00 El BSA 80 00 70 00 60 00 Sample ID 50 00 40 00 30 00 Sample 9 10 mm Absorbance 20 00 A fA dE 280 Gees 24 412 10 00 A 280 10 mm path 24 412 0 00 260 280 0 66 0 0 ER 1 1 1 H U H H 220 230 240 250 260 270 280 290 300 310 320 330 340 350 mg ml 36 60 Wavelength nm 3 5 1 B B2545 0 02 48 16 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 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 Proteins A280 File Edit Help M Re blank Recordinc Measurement complete 6 14 2007 1 18PM easure Print Repot 1 Show Report User Default Overlay control Accumulate until clear v 340 nm normalization 7 On SampleType 1342 1 BSA 12 00 TN X 10 00 Sample ID 6 00 Sample 3 4 00 P o D Sa o i A 280 Abs 0 966 A 280 10mm path 0 966 260 280 0 79 zi Hi i 1 1 1 H D D D H 220 230 240 250 260 270 280 290
29. 300 310 320 330 340 350 mg ml 145 Wavelength nm 3 5 1 B BB710 1 39 96 24 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 does not clear the existing overlaid sample spectra 8 2 Section 9 8BProteins 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 metalloproteins 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 NanoDrop 1000 Spectrophotometer 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 fo
30. 50 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 absorbance 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 Beer s 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 apprec
31. 550 0 04 0 05 Below e D 2 50E 5 650 0 00 0 05 J9 Delete Alexa Fluor 546 f Selected Alexa Fluor 555 Alexa Fluor 594 it Alexa Fluor 647 Selected Alexa Fluor 660 035 055 Note predefined dyes are indicated with a diamond and cannot be modified Sample Volume Requirements Field experience has indicated 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 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 Reproducibility 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 sample ran
32. 66 7 QA uM 0 00 1 00 1 1 i 1 1 1 220250 300 350 400 450 500 550 600 650 700 750 Wavelenath nm 35 1 B BB710 1 39 96 24 Absratio alas 112 mgimL 2 6 9 1 Section 9 8BProteins amp Labels 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 3 user selected wavelength Norm Abs A3 10mm equivalent normalized absorbance at the respective wavelength Dye 1 or 2 user selected dye Norm Abs X1 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 21 43 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 no
33. 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 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 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 Protein standards BSA for generating a standard curve may also be provided by the manufacturer for the BCA assay kit 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 NanoDrop 1000 Spectrophotometer measures higher protein concentrations than traditional cuvette based spectrophotometers you ma
34. DP 4 1 COMMO FUNCIONS EH 4 1 Measte KREE 4 1 BIEN EE 4 1 ReDIanK EE 4 1 mulis pim cc 4 2 Stat Report RECON ERE ES 4 2 PRM REDON RE 4 2 SHOW REDON E E 4 2 Sample pe PES 4 2 EEN 4 2 oH EE 4 2 Escape Key EE 4 3 Show Context Help CAHEN 4 3 Users Oe E EE 4 3 Nuclei E 5 1 Sample Volume Heouremente nnns 5 1 Measurement Concentration Hange 5 1 Spectrum Normalization ccccccccsesseeececeeeceeeeeeeeeeeeseeeeeeeeeeesseaeeeeeeeeees 5 1 Spectrum Overlay Control 5 2 MICE OAV AY em E 6 1 Fluorescent Dye Gelechon eiin aeneae aeai aaeai 6 1 Sample Volume Heouremente nnns 6 1 Measurement Concentration Hange sese 6 1 Baseline Calculation amp Normaltzatnon 6 1 BCEE 7 1 Sample Volume Requirements nnne 7 1 Measurement Concentration Hange 7 1 Unique Screen Features 7 1 Protein Deeg aiai 8 1 Sample Volume Requirements nnne 8 1 Pedestal ECONO MING EEN 8 1 Measurement Concentration Hange 8 1 lJnique Screer RE 8 1 Spectrum Normalization ccccccccssseecccceeeceeeeseeeeceeeseeeeeeeeeeeesseaeeeeeeeeees 8 2 Spectrum Overlay Control 8 2 Proteins WE E 9 1 Fluorescent Dye Gelechon a E a 9 1 Sample Volume Requirements nnns 9 1 Pedestal HeconditlohlliQ BEE 9 1 Measurement Concentration Hange sess 9 1 Billie UC RE 9 1 BASCIING EE 9 2 Protein BCA EE 10 1 Sample Volume Requirements ns 10 1 Pedestal Reconditioning EE 10 1 Measurement Concentration Hange 10 1
35. Ee 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 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 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 NanoDrop 1000 Spectrophotometer 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 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 1 M cm nm 280nm amp 280nm 4 TEES 03 1 50E 5
36. File Folder 10 15 2004 10 50 AM C3 BCA Protein uy vis File Folder 10 15 2004 10 50 AM Bradford Protein 4 280 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 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 operating software that offers the user the ability to customize report structures import stored data and re plot data from previously generated data 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 A NanoDrop 1000 spectrophotometer does not need to be connected to the PC to use the Data Viewer module Data Viewer Fea
37. HiAbs 7 On Sample ID FITC Sample Baseline A1 0 008 D 20 i i 1 220 250 300 350 400 450 500 3 5 1 8 82545 0 11 48 16 Wavelength nm 1 Abs1 and A2 Abs2 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 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 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 NanoDrop 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
38. Lowry assay is run Although curves can be saved and reloaded in the NanoDrop 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 up to 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 ile Edit Standard Curves Help Re biank Print Screen Recording on Blank Pim Aapon Show Repon Stnderd Ouve Measurement Type Standards missi ac Step 1 Measure the Reference Lowry reagent a zero Standard 1 Standard Note The software will guide the user with instructions in the large text box on
39. Te ec S extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution 8 1 Section 8 Protein A280 User entered values for molar extinction coefficient M cm and molecular weight MW in kilo Daltons for their respective protein reference Maximum value for e is 9999 X 1000 and maximum MW KDa soon value for M W is 99999 X 1000 Other protein 1 NP User entered mass extinction coefficient L gm cm 1 for a 10 mg ml 1 solution of the GEES 0640 respective reference protein 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 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 Proteins A280 File Edit Help Re blank Recording dl Measurement complete 6 15 2007 1
40. ally use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the NanoDrop 1000 can measure higher protein concentrations than traditional cuvette based spectrophotometers 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 0 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 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 unknown ee ee Making Lowry Measurements A standard curve is required every time the Modified
41. andard Curve Type Interpolation 1 mm Absorbance p5 i D 1 DH H 000 050 100 150 200 250 BCA Protein Std Curve Eile Show Context Help Reference mg ml Ave Abs 0 000 0 003 Abs 1 0 004 Abs 2 0 003 350 400 4 mg ml Abs 3 0 002 0 550 600 650 700 750 800 Standard 1 0 013 0 025 0 050 0010 0 008 0 065 0 010 0028 0 065 0 010 0027 0 065 0 009 002 0 064 User Default 6 5 2007 3 31 PM 351 4 B2545 0 03 48 24 0100 0200 0 148 0 279 0 146 0276 0 148 0278 0 148 0 280 1 mm Absorbance R squered 0 9999 Standard Curve Type interpolation 0 05 U H H U U H U Uu Uu U U H U H 000 001 0 02 0 03 0 04 0 05 0 06 0 07 008 0 09 0 10 0 11 012 013 0 14 0 15 0 16 0 17 0 18 0 19 020 mg ml Regular BCA Standard Curve 0 2 8 0 mg ml mini BCA Standard Curve 0 01 0 20 mg ml Section 10 9BProtein BCA It is recommended that you process all of the unknowns to be assayed before exiting the BCA software module 10 3 Section 11 Protein Lowry 11 Protein Lowry The Modified Lowry Protein Assay is an alternative method for determining protein concentration based on the widely used and cited Lowry procedure for protein quantitation Like the BCA and Bradford Assays the Modified Lowry Assay requires standard cu
42. aved 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 s 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 Chote mis optan to be able to reload the Full Report repag sto the Data Witwer at amp labor dene Export Report Choose this option bo expo tab daliminmted po po inv file of the dispered Asport Table Table Only suitable for impar into Pce Choose this option bo apan tab delimimted Export Report amp ioxifie ofthe displayed Report amp Stenderds Standards Tables Tables suitnbla tor import ina Excel Cancel Using the Full Report option will allow the user to use the Data Viewer to reload the report at a later date The saved report may be recalled using the pull down Load Report If using the Load Report feature the report will be displayed with the default column configuration Note Access the User Preferences module on the main menu
43. bsorbance in the UV range such as protein or nucleic acid has dried down on the pedestal Follow the cleaning steps outlined below 1 Apply 5 ul of dH20 solution onto each bottom pedestal 2 Lower the upper pedestal arm to form a liquid column let it sit for approximately 2 3 minutes 3 Wipe away the water from both the upper and lower pedestal with a clean lab wipe Note Typically dH50 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 the dH20 in step 1 above Follow with 5 ul of dH20 to remove any residual HCI Note Do not use a squirt bottle to apply de ionized water or HCI 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 17 2 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
44. ce using the Reset all Standards button Protein Bradford Std Curve Ele Show Context Help ug ml AveAbs Abs 1 Absorbance Abs 2 Abs 4 Abs S 4 User Deteult 6 12 2007 2 07 PM Reterence l 0 022 0018 Standard 1 2 0 041 0 042 Standard 2 2 0 065 0 069 Standard 3 j 0101 0 100 0021 002 0 044 0 066 0102 0 100 0025 2 0038 64 0060 0 098 0 022 ag 3514 A152 1 35 48 24 0 067 0104 Standard 4 0122 0 122 Standard 5 Standard 6 Standard 7 0 125 20 0 125 ne R squared Standard Curve Type 2nd Order Polynomial v Protein Bradford Std Curve Ele Show Context Help ug ml AveAbs Abs D 45 50 55 60 Absorbance Abs 2 ug ml Abs 3 Abs 4 Abs 5 4 65 70 75 80 85 90 95 100 User Default 6 12 2007 2 07 PM Reference I e2 0018 Standard 1 2 0 042 Standard 2 2 0 069 Standard 3_ 0101 0100 0021 0 044 0 066 0102 0 022 0 042 0 064 0 100 0025 0038 0060 351 4 A152 1 36 48 24 Stenderd4 100 22 0122 Standard 5 Standard 6 Standard 7 0 125 0 120 0125 0098 010 Standard Curve Type 2nd Order Polynomial ze i 1 45 50 ug ml 55 60 65 70 75 80 85 90 95 100 Exiting the Bradford Module It is recommended that you process al
45. cing 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 in the following image bod de Corni or CS bey to tee mole Import amp Return 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 Directory tree Used to select specific data to be imported Clicking on the square to the left of each file name will provide further detail to each level Users may choose to select either individual samples within a file or the entire file All import selections must be of the same application or method type Note Hi Abs data cannot be imported into the Data Viewer but can be opened with Excel type spreadsheets Archive File Converter Data generated with earlier versions of the operating software and not stored in c nanodrop data will need to be copied and converted to 3 2 version compatible ndj files See Archive File Converter later in this section for a more detailed explanation of this feature Note The original archive files will not be altered
46. cleic 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 19 1 Section 19 Appendices 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 the NanoDrop 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 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 1000 Spectrophotometer is compatible with most solvents typically used in life science lab
47. confirm that both pathlengths are within calibration specifications Utilities amp Diagnostics Bii sas Calibration Return to 3 Check Main Menu A vial of CF 1 is required to run the calibration check procedure CF 1 is an aqueous potassium dichromate K2Cr2O7 solution for use in confirming calibration of NanoDrop 1000 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 Thermo Fisher Scientific or your local distributor Procedure for Removal of Lint Build up Under Solenoid On occasion the top and bottom pedestals may require reconditioning which involves rubbing the pedestals aggressively 30 40 times with a lab wipe Some brands of lab wipes shred during the process and may result in a build up of lint under the instrument solenoid A significant build up of lint may alter the absorbance pathlength resulting in erroneous measurements Refer to the image and follow the instructions below to remove any lint build up prior to performing the calibration check 1 Lay the instrument on its side with the source fiber black fiber optic cable facing up 2 Open the arm of the sampling mechanism 3 Using a paperclip or a small screwdriver manually depress the solenoid plunger and spray compressed air down the solenoid plunger hole Be sure to keep the can of compressed air upright so as not to
48. d 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 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 in 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 val
49. ded that both measurement pedestals be cleaned with de ionized water upon completion of a testing series 1 Apply 5 ul of dH20 solution onto each bottom pedestal 2 Lower the upper pedestal arm to form a liquid column let it sit for approximately 2 3 minutes 3 Wipe away the water from both the upper and lower pedestal with a clean lab wipe Note Typically dH50 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 the 5 ul of dH20 Please follow an HCI application with 5 ul of dH20 to ensure any residual HCl is removed Note Do not use a squirt bottle to apply de ionized water or HCI Calibration Pathlength Accuracy Calibration Check It is good practice to verify the calibration every six months using CF 1 calibration fluid This will verify the pathlength accuracy of the instrument The calibration check procedure is found within the Utilities and Diagnostics module Wavelength Each time the software is started the wavelength is auto calibrated based on known peaks in the xenon lamp spectra No calibration is required by the user Parts That Require Replacement In general the xenon flash lamp is the only part that will need to be replaced The lamp has a lifespan of at least 30 000 measurements When the flash lamp fails the light output will become v
50. e 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 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 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 whic
51. e by selecting Start gt Programs NanoDrop 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 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 9000 This error occurs when the passwords log file is missing or corrupt Reinstall the operating software and overwrite the existing copy when prompted A new copy of the passwords log file should appear in the CANanoDrop Data og Files folder 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 y
52. e 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 flash lamp is functioning Wisible Sporn Au LAR Seeche Sonal Humber UE bU Esq Configuration 1 s 05 ora Dodecinr Bias 273 r Wavelength Arrunmey Mex Single Ofset 044 anon Peaks Monitored 4B4 nm 4839 SHO 7 nm ena 25m 3525 eae 8211 A i i D D i eeu 3000 300 0 500 0 Bin o 700 0 500 0 8507 Minentenath pm f 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 solves the problem contact your local distributor or Technical Support UV Wavelength signal Intensity is low UY wavelength signal intensity is low Cancel The above error message indicates that the detector signal in the UV region is low The most likely explanation for the signal is that a sample with a
53. e selected scheme r Power schemes Save As Delete r Settings for Home Office Desk power scheme When computer is Plugged in Running on batteries Turn off monitor Atter 20 mins DI Atte 15 mins DI Turn off hard disks Never After 10 mins DI System standby Never b After 30 mins m System hibernates Never Mw After 45 mins ind Cancel Static Electricity Discharge Discharge from the user to the instrument can be a problem in very dry environments It may be necessary for the user to wear a grounding strap to prevent the discharges from occurring Defective USB Port on PC 17 1 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 12V power supply For mare details referto the Troubleshooting section of the User s Manual available from Main Menu 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 th
54. ears 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 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 Bradford reagent only no protein and a single replicate of one standard The multi point standard curve generator allows a maximum of five replicates for up to 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 lt is advisable to use water as the blank and use the dye reagent without any protein added as t
55. ecules such as genomic or lambda DNA are particularly susceptible to this phenomenon Note Larger volumes used with cuvette based spectrophotometers will 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 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 Some buffer components 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 Measuring samples at or near the detection limit will result in measurements that can vary a significant amount Refer to the Measur
56. ed in system software compatible with Windows 2000 or XP Blanking and Absorbance Calculations When the NanoDrop 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 Calculation 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 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 Nu
57. ee orders of magnitude see our website for sample data 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 1 296 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 advantages 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 pos
58. ement Concentration Range of the application module that you are using for the applicable measurement range Confirm instrument accuracy with CF 1 CF 1 is a concentrated potassium dichromate calibration standard available from Thermo Fisher Scientific and its distributors It is 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 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 1000 Spectrophotometer Spectrophotometer to other spectrophotometers it is important to ensure that the pH of an undiluted sample measured on the NanoDrop 1000 Spectrophotometer 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 steep
59. ery erratic or stop altogether Contact Technical Support or your local distributor if you suspect your lamp may need replacing Warranty All NanoDrop spectrophotometers and accessories manufactured by Thermo Fisher Scientific 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 More information about the various plans may be found on our website Section 19 Appendices 19 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 Range 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 Includ
60. following 1 Confirm that the USB cable is connected to both the PC and the instrument 2 Disconnect and then reconnect the USB cable If the Found New Hardware Wizard appears follow the prompts for automatic installation of the software drivers 3 Restart the operating software if it works properly you are finished If it does not operate properly go to step 4 4 Locate the My Computer or Computer icon on the desktop or access through the Windows Start menu and right click to open 5 Highlight and select Manage 6 Click on Device Manager in the left pane to populate the right pane with the connected devices 7 Locate Thermo Scientific NanoDrop in the right pane and click on the sign to open A yellow exclamation point or question mark next to NanoDrop 3300 will indicate improper driver installation Note f a Thermo Scientific NanoDrop device is not listed in the Device Manager search the entire device list for any device with a yellow flag The device may be listed as an unknown device a generic device a NI Visa device or may be included in the Universal Serial Bus controllers listing 8 Highlight the yellow flagged device and right click to uninstall the device 9 Disconnect USB and power supply wait 5 seconds and reconnect starting with the power cord first 10 Try a second USB port or install the instrument on another PC to rule out a faulty USB hub port on the original PC If none of the troubleshooting steps above solves
61. g a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning oolutions and reagents containing surfactants may 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 on our website Measurement Concentration Range The NanoDrop 1000 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 read RRE minimum 5 replicates SD mg ml CVz Purified BSA 0 10 mg ml 100 mg ml sample range 0 10 10 mg ml 0 10 mg ml l sample range 10mg ml 2 Unique Screen Features File Edit Help Re blank Measurement complete 6 14 2007 1 07 PM Measure Exit Print Repot 1 Show Report Use
62. ge gt 8 0 pmol ul 2 sample range 0 30 6 0 pmol ul Alexa Fluor 546 0 30 145 0 30 pmol ul sample range gt 6 0 pmol ul 2 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 750 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 6 1 Section 6 5BMicroArra Unique Screen Features MicroArray File Edit Help Re blank Print Screen z c e 6 14 2007 1 52 PM weCOruIng Show Report Default 056 Max Absorbance Sample Type ssDNA 33 l Sample ID Microarray sample Sample 3 A 260 gt AbsNorm 0 175 Dye 1 Gi v Abs 0 027 18 Nani pmol ul Dye 2 Cys a Abs 0513 pmol ul 20 5 Norm 3067 e e i i r 260 280 1 80 220250 300 350 400 450 500 550 600 650 700 750 Wavelength nm ng ul 54 9 3 5 1 B 82545 0 02 56 16 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 1
63. h 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 ND 1000 Data Viewer vi Die Configuration Data Repons Help Ignore 6 20 2007 51 Ah Repart Name Report Full Mode TE Print P MaxReportSize 200 d Prnt Semple Date Time nod A260 A280 260 280 260 230 Constant Cursor Cursor 340 Measurement A ID Pos abs raw Type 5 Le 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 15 3 Section 15 Archived Data and Data Viewer Rapert condiguration editar sulect The report columns to display and the order Triad the columns should appear Change the order by selecting and dragging a Cham header sting to the deoze ed poston Report columns Show All Move Up _Move Down Delete 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 Save Report Format Saves the current report format as an ndf file for retrieval and future use To designate a s
64. he 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 Ele Edit Standard Curves Help Re blank f PrintScreen Recording View Stenderd Curve Measurement Type Measure up to 5 replicates of the reference Use 2 ul samples e user ma i P i lt Standards run a new Blank at anytime if needed Reference and Standards os een LCENNUMIE REN 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 Cursor B 72 2 Absorbance 9 595 nm 0 000 MGE M ugimi oy gros Step 2 Measure Standards get f Pr Repon ShowRepon Seed Cuve et Measurement Type z Sample Standards Up to 5 replicates of each of up to 7 standards can Reference and Standards be measured The software will not allow measurement of samples until a minimum of 1 standard and Si references or 2 standards are measured ENE Polynomial curve fitting requires more standard Cursor TEE points Absorbance Absorbance 595nm 0140 0 05 r 575 600 625 650 675 ugiml 2103 351 82545 003 48 24 Wevelengh nm Ele Edit Standard Curves Help Rebiank f PrintScreen Recording View 6 14 2007 1 32 PM ie coral ie Show Report f _ Standard Curve ser Deteutt
65. hing 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 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 4 2 Section 4 Common Module Functions 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 applicatio
66. i cett 3000 400 0 9m BOU a d DUU 950 VWienwnlangth nm 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 local distributor or Technical Support Data Archive Files If you have questions about your data please send the archive file containing the suspect data as an email attachment to your local distributor or Technical Support The archived file can be found at C NanoDrop 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 17 6 Section 18 17BMaintenance and Warranty 18 Maintenance and Warranty Cleaning The primary maintenance requirement of the NanoDrop 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 recommen
67. iably 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 in 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 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 individual 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 NanoDrop 1000 spectrophotometers will measure absorbance up to the 10mm pathlength equivalent of 15 A NanoDrop 1000 Spectrophotometer instruments with serial numbers gt 500 or those that have been field retrofitted can utilize the short path length 0 2mm which enables the 10mm path length equivalent of 75 A Unique Screen Features UV Vis Module File Edit Help Re blank Recording Measurement complete 6 14 2007 12 59 PM e xit Print Report Show Report User Default Mex Absorbance Normalize V On
68. ierce 660 nm Bile Edt Standard Curves Help Step 1 Measure the Reference 660 nm reagent a Measurement Type Zero Standard Standards Reterence ond Standards Conc Ave Abs Note The software will guide the user with instructions in Stenderd the large text box on the right side of the screen Absorbance Absorbance 9 660 nm 0 000 05 e e e e d 550560 560 600 620 640 660 680 700 720 740750 Wavel n ug ml NaN 36181531 021 80 16 avelengh nm Ble Edt Standard Curves Help Step 2 Measure Standards Measurement Type xaid Cun Sample Stendards D E m Up to 5 replicates of each of up to 7 standards can be measured Standard Senda 7 60 The software will not allow measurement of samples until Ka mamm a minimum of 1 standard and references or 2 standards are measured Polynomial curve fitting requires more standard points Absorbance 660nm 027 0 05 i D i j 1 4 550560 580 600 620 Ge 660 700 20 40750 ug ml 2000 3618153 0 21 80 16 nm Eile Edt Standard Curves Help SR View 7 17 2008 10 20 AM Punt Repor Standard Curve SC Measurement Type Load 2 ul of your sample and dic the measure button Semple Standards Step 3 M eas u re Sam p es Sample ID Sample 1 040 Max Absorbance Sample concentrations can be calculated by using linear interpolation point to point between the two standards flanking the unknown sample
69. int Report A Show Report User Default Overlay control Clear graph each Sample Sample Type DNASD Sample ID dsDNA Sample A 230 inm Abs 17 554 A 260 10 mm path 39 019 A 280 10 mm path 20 576 260 280 1 90 260 230 2 22 4 30 1 I I D I i i 1 1 1 1 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm ng uL 19509 3 5 1 B BB708 0 78 128 16 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 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 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 an
70. iquid 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 on our website Measurement Concentration Range The NanoDrop 1000 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 Approx Typical Reproducibility pde uoa Upper minimum 5 replicates yp EI SD mg ml CV Purified BSA 0 10 mg ml 20 20 mg ml sample range 0 10 10 mg ml 0 10 mg ml sample range 10mg ml 2 sample range 0 20 4 0 pmol ul 0 20 pmol ul sample range gt 4 0 pmol ul 2 Unique Screen Features Proteins amp Labels File Edit Help 5 14 2007 1 12 PM User Default Max Absorbance 10 00 Baseline Type 750 nm v 340Bichrometic Mon Sample ID 10 00 9 00 SampleType d IgG 8 00 7 00 6 00 5 00 Sample 400 AS 200 Norm Abs 9a3 4034 10 mm equivalent absorbances a o B o E E e 3 00 2 00 Dye 1 FITC FAM v Norm Abs 4 4538
71. ition start the operating software by selecting the following path Start gt Programs gt NanoDrop gt ND 1000 version E NanoDrop 1000 3 7 File Hep Default Lites amp Diagnostics Dye Chromo phore Editor Account Management Application Modules The operating 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 3 2 Section 3 General Operation 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 e Pierce 660 nm Protein Assay protein concentration using the new 660 nm 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 In addition to the primary data storage of archive files at c nanodrop da
72. l of the unknowns to be assayed before exiting the Bradford software module 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 12 3 Section 13 Protein Pierce 660 nm 13 Protein Pierce 660 nm The Thermo Scientific Pierce 660 nm Protein Assay reagent is a ready to use formulation that offers rapid accurate and reproducible colorimetric detection of minute amounts of protein in solution The reagent is ideal for measuring total protein concentration in samples containing both reducing agents and detergents Dynamic Range The assay has a linear range for BSA of 50 2000 ug ml using a reagent to sample ratio of 15 1 The sensitivity of the assay may be increased by using a 7 5 to 1 reagent to sample ratio yielding a linear range of 25 1000 ug ml Aca Approx Approx Typical Reproducibility T Hd Lower Upper minimum 5 replicates yp Limit Limit SD mg ml CV 50 ug ml 2000 ug ml 5 over entire range 25 ug ml 1000 ug ml 5 over entire range e A regular assay using a 15 1 reagent sample volume ratio To accurately prepare standards we suggest using a minimum sample volume of 4 ul in 60 ul of the Pierce 660 nm reagent larger sample volume is preferable e A mini assay using a 7 5 1 reagent sample volume ratio To prepare sufficient volume of mixtures we suggest using a minimum of 6 ul of sample and 45 ul of
73. lowing 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 on our website 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 3 1 Section 3 General Operation 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 NanoDrop 1000 Spectrophotometer 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 thr
74. ly 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 17 5 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 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 SH 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
75. module 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 7 1 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 ready 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 properties in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter the surface tension resulting is difficulty in forming adequate columns for measurement The column formation issue can be overcome without affecting the sample s absorbance by usin
76. mple 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 on our website Measurement Concentration Range The Modified Lowry assay concentration range of detection is 0 20 mg ml to 8 0 mg ml BSA on the NanoDrop 8000 Approx Approx Typical Reproducibility Assay Lower Upper minimum 5 replicates Type Limit Limit CV 96 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 Protein standards BSA for generating a standard curve may also be provided by the manufacturer for the Modified Lowry assay kit Follow the manufacturer s protocol for the assay including recommended incubation times and temperature Addition
77. mple or 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 gt s e c I ce 1 1 i EEG i 1 mm Absorbance Absorbance 9 B50nm 0 082 0 05 D D H 1 D D D D D 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 mg ml 1 809 Wavelength nm 35 1 8 88708 0 96 128 16 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 Software versions 3 5 1 and higher include 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 T Protein Lowry Std Curve Eile Show Context Help Absorbance User Deteult Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 6 4 2007 4 48 PM Re
78. n modules It can also be accessed by selecting from the Help pull down menu in any application module or from Start 2 Programs gt NanoDrop ND 1000 version 4 3 Section 5 4BNucleic Acids 5 Nucleic Acids Nucleic acid samples can be readily checked for concentration and quality using the NanoDrop 1000 Spectrophotometer To measure nucleic acid samples select the Nucleic Acid application module 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 1000 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 296 Unique Screen Features Nucleic Acids E File Edit Help Re blank A Print Screen Measurement complete 6 14 2007 1 13PM l Measure Exit Pr
79. n any calculations ug ml concentration of the sample unknown Making Pierce 660 nm Protein Measurements A standard curve is required by the software every time the Pirece Protein 660 nm assay is run Although curves can be saved and reloaded in the NanoDrop 1000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines regarding the use of saved curves when running this 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 reagent only no protein and a single replicate of one standard The multi point standard curve generator allows a maximum of five replicates for up to 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 use the dye reagent without any protein added as both the Blank and the 0 reference sample Note This is unlike the other colorimetric assays on the NanoDrop 1000 where it is recommended that water be used as the blank There are three general procedural steps to unknown protein concentration measurements The requisite order including generating the standard curve is as follows Section 13 Protein P
80. nsure 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 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 Pedestal Reconditioning The Bradford reagent as well as other buffers containing surfactants can un condition the measurement pedestal surfaces so that the liquid column does not form well with 1ul samples If this occurs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface al
81. or 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 Sample Absorbance Absorbance 660mm 0199 0 04 SN D D D 1 i D D D i 550560 580 600 620 ye Se seg 680 700 720 740750 ug ml 1465 36 1 BLab 0 52 128 32 a 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 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 Eile Show Context Help Delete Point Absorbance User Detault ug ml AveAbs Abs Abs 2 Abs 3 Abs 4 Abs 5 7 17 2008 10 08 AM 003 0000 0003 J02 c Standard curve dats E 33 0035 0033 361 BLab 052 128 32 The 15 1 reagent to sample Fesquered 0 9983 Standerd Curve Type inear ratio assay covers 900 2000 ug ml
82. oratories 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 HNOS 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 0 596 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 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 the NanoDrop 1000 Spectrophotometer 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 ad
83. ords 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 WanoDrop Data Log 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 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 96 field Refer to the dye manufacturer to find the 260 nm and 280 nm factor for dyes not pre defined in the Dye Chromophore List Note If upgrading from a version p
84. our local distributor or Technical Support 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 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 on a computer running Windows 2000 or XP It occurs because the user does not have the necessary authorization to install the software Contact your system administrator if this occurs Insufficient Memory This error message or others with similar wording occurs when attempting to install the operating software on a computer that does not have at least 40MB of free hard disk space No Printer Connected This error appears when attempting to print when a printer is not attached to the PC It is non fatal and will not cause the software to shut down 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
85. pend the cells when sampling for absorbance measurements and read the sample immediately to avoid significant cell settling Vigorous mixing may be required 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 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 14 1 Section 15 Archived Data and Data Viewer 15 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 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
86. properly and that the pedestal is clean Follow the steps below to perform a blanking cycle 1 Load a blank sample the buffer solvent or carrier 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 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 4 1 Section 4 Common Module Functions Print Screen F4 The Print Screen button will print a copy of the current operating screen to the default printer attached to the operating PC Note The system i
87. r Default Overlay control Clear graph each Sample 340 nm normalization 7 On SampleType B T X Sample ID Sample 2 m o o a o S e A 280 10 mm path 260 280 33 GEN r 1 1 1 1 1 H 220 230 240 250 260 270 280 290 300 310 320 330 340 350 mg ml 1 43 Wavelength nm 3 5 1 8 BB710 1 39 96 24 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 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 A description of each sample type is given below TAbs Tmgiml 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 BEA Bovine Serum Albumin reference Unknown sample protein concentrations are calculated using i the mass extinction coefficient of 6 7 at 280 nm for a 196 10 mg ml BSA solution al laG IgG reference Unknown sample protein concentrations are calculated using the mass extinction i coefficient of 13 7 at 280 nm for a 1 10 mg ml IgG solution Lysozyme Lysozyme reference Unknown sample protein concentrations are calculated using the mass
88. r measurement and concentration calculation The default settings 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 Name en nm 260nm 280nm fa TE 1 50E 5 550 0 04 0 05 Below 250E 5 650 0 00 0 05 Delete Alexa Fluor 546 04E S Selected Alexa Fluor 555 Alexa Fluor 594 30E 1 Alexa Fluor 547 Selected Alexa Fluor 660 Cy3 5 Cy5 5 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 variable 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 oolutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the l
89. rior to 3 3 zero values 0 for 260 nm 96 and 280 nm correction factors will be entered for all user defined dyes Dye Chromophore List Editor Dye Chromophore List Name Cy3 Cy5 2 50E 5 Insert IRIG 0 05 Below Alexa Fluor 488 7 10E 4 0 0 Alexa Fluor 546 104E 5 D j Selected Alexa Fluor 555 1 50E 5 Alexa Fluor 594 7 30E 4 Alexa Fluor 647 2 39E 5 Alexa Fluor 660 1 32E 5 035 1 50E 5 e Cy5 5 250E 5 DyLight 488 7 00E 4 DyLight 549 1 50E 5 DyLight 594 8 00E 4 DyLight 633 1 70E 5 827 e DyLight 649 250E 5 6 4 Mil inht RAN 1 4NF 5 Selected Note predefined dyes are indicated with a diamond and cannot be modified 3 5 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 C For best results ensure measurement pedestal surfaces are clean load a water sample onto the lower measurement pedestal and then click 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 File Edit Help Make new BLANK 6 4 2007 4 00 PM
90. rmalized at 340 nm See the Concentration Calculation Beer s 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 The user may also elect whether or not to use a bichromatic normalization of the 280 nm absorbance value to the absorbance value at 340 nm 9 2 Section 10 9BProtein 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 its wavelength maximum of 562 nm and normalized at 750 nm Pre formulated reagents of BCA and CuSQ 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
91. rve 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 Solutions and reagents containing surfactants may 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 sa
92. s 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 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 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 Data Viewer in Section 14 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 ND 1000 Data Viewer vi File Configuration Deta Repors Help Ignore 6 20 2007 10 21 A Testtvpe E 5 20 2007 10 21 AM Plots Report Ma Seve Report Name Report Full Mode Base rint Sample Date Time ng u A260 A280 260 280 260 230 and Prir MexReportSize 200 7 ar it Constan t f Cursor Cursor 340 Measurement 4 Pos abs raw Type All data is stored in
93. sed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light after passing through the sample The instrument is controlled by PC based software 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 1000 Spectrophotometer The small sample requirement and ease of use make the NanoDrop 1000 Spectrophotometer ideally suited for measuring 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 e Bradford Assay analysis of protein e BCA Assay analysis of protein e Lowry Assay analysis of protein Pierce Protein 660 nm Protein Assay Cell density measurements e General UV Vis spectrophotometry Patents The sample retention technology used in the NanoDrop 1000 Spectrophotometer 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 operating software will only run on an IBM compatible PC meeting the below criteria No Mac versions of the software are currently available Compatible with Windows 2000 XP Visa
94. sible solutions Configuring the System Font The 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 Choosing an alternative font may result in some text being truncated in the operating software window Software Upgrades Periodic upgrades are made to the operating software and are available for download See our website for the latest available software version 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 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 flash lamp is not energized Also the instrument does not utilize a power switch or give a 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
95. suring 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 oolutions and reagents containing surfactants may 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 on our website Measurement Concentration Range Unknown protein concentrations from 100ug mI up to several thousand micrograms ml ug ml can be determined on the NanoDrop 1000 Spectrophotometer using the regular Bradford assay The best linearity is in the 100 1000
96. t 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 Use the same pipettor for both volumes to eliminate any pipette to pipette accuracy differences Protein standards BSA for generating a standard curve may also be provided by the manufacturer for the Bradford assay kit 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 NanoDrop 1000 can measure higher protein concentrations than traditional cuvette based spectrophotometers 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 0 sample Replicate counter for tracking replicate number during reference and standard measurement Reset all Standards F11 clears all replicates of all standards Section 12 11BProtein Bradford Reset 1 Standard F12 cl
97. t 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 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 remove 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 is clean of black residue To check the effectiveness of the reconditioning load a 1 ul aliquot of dH50 onto the lower measurement pedestals and visually verify that the water beads up Additional information about the PR 1 kit may be found on our website 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
98. ta 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 the Auto Reporting feature which allows data to automatically populate the report for all samples To enable this feature select the Reports tab of Users Preferences and select the box next to each application listed in the Auto Reporting area Save these preferences by selecting Save amp Exit to close the 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 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 wa
99. ted Note The actual 1 mm absorbance is displayed A 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 is completely covered by sample Cell Suspension Concentrations Due to its shorter path length the NanoDrop 1000 Spectrophotometer can measure absorbencies that are 10 fold higher than those 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 Sample Homogeneity The user must be sure to homogeneously sus
100. terence 0 009 0 010 0 011 Standard 1 0000 0003 0000 LET A BENS 011 40 24 Standard 2 0016 0016 0017 0016 Standard 3 0025 0025 0025 Standard 4 0031 0032 0033 Standard 5 0104 0098 0105 n oT Modified Lowry Resquered 03960 Stenderd Curve Type Standard Curve 0 2 4 0 mg ml mg ml Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module Section 12 11BProtein Bradford 12 Protein Bradford The Bradford Assay is a commonly used method for determining protein concentration It is often used for more dilute protein solutions where lower detection sensitivity is needed and or in the presence of components that also have significant UV 280 nm absorbance Like the BCA and Lowry methods the Bradford assay requires a standard curve 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 en
101. the Pierce 660 nm reagent in a PCR tube 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 NanoDrop 1000 can measure higher protein concentrations than traditional cuvette based spectrophotometers 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 0 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 660 nm the protein reagent complex s absorbance at 660 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 i
102. the archive file at C NanoDrop Data and in a duplicate location if selected in User Preferences 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 how the buffer is handled Refer to Data Viewer in Section 14 for additional information All data is stored in the archive file at C NanoDrop 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 out 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 flas
103. the right side of the screen 1 mm Absorbance Reset All Standards Delete 1 Standard Cursor Absorbance 9 B50nm 0 000 TA 435 ato dr soo sis sio s s edo vie eso GidSamplespecrun Semple spectrum 400 425 450 475 500 525 550 575 600 625 650 67 p p mg ml NaN 351 85342 043 96 24 Wavelength nm Measurement Type Measure up to 5 replicates of the standard Use 2 ul samples The user may P a mae P m Sample Standards ew Blank at anytime if needed Reference and Standards Step 2 Measure Standards 050 gt Max Absorbance Up to 5 replicates of each of up to 7 standards can be measured Standard 4 1 000 Standard 6 3 000 Standard 7 4000 The software will not allow measurement of samples until a mesasa ec minimum of 1 standard and references or 2 standards are Cursor EE measured Polynomial curve fitting requires more standard points Absorbance Absorbance 650nm 0 078 0 05 i Sa spectru 400 425 450 475 500 525 550 575 600 625 650 675 700 7 U mg ml 2 000 351 88708 0 96 128 16 Wales nm Ele Edit Standard Curves Help Re blank f PrintScreen Recording View 6 14 2007 3 47 PM Show Report j Stenderd Curve ser Desch Measurement Type Sample Standards Sempie10 Lowy Step 3 Measure Samples 050 Max Absorbance Sample Sample concentrations can be calculated by using linear interpolation point to point between the two standards flanking the unknown sa
104. to try the following to ensure representative results Make sure the sample surfaces are clean before starting the software module 17 4 Section 17 Troubleshooting A dirty sample pedestal i e a pedestal with a sample dried on it 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 before opening an acquisition module to remove any dried down 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 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 NanoDrop 1000 Spectrophotometer it is important to ensure that the sample being measured is homogeneous Field experience has shown that samples containing large mol
105. tures 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 15 1 Section 15 Archived Data and Data Viewer 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 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 report See Reports later in this section for a more detailed explanation of the features of the Report Page 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 pla
106. ue of the sample at 340 nm which should be very nearly zero absorbance All spectra are referenced off of this zero 5 1 Section 5 4BNucleic Acids 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 Nucleic Acids Eile Edit Help M Re blank PrintScreen Recording F Measurement complete 6 14 2007 1 30 PM easure Print Report 1 Show Report User Default Overlay control Accumulate until clear v Sample Type DN amp 0 Sample ID Sample ki 230 z nm Abs A 260 10 mm path A 280 10 mm path 260 280 260 230 2 27 5 31 1 H I H I I I I 1 1 1 J 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm ng uL 11857 3 5 1 B BB708 0 78 128 16 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 5 2
107. ution is collected in the bottom portion of the ampoule 7 Carefully break the neck of the ampoule to open the CF 1 Calibration Fluid 8 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 9 Ifthe instrument does not pass the calibration check using 1ul samples immediately repeat the procedure again step 6 using 2ul samples A table of the Calibration Check tolerances and specifications is available through the Help menu drop down 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 your local distributor or Technical Support 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 EE 17 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
108. vanced printing features box is checked Click on Printing Defaults then Advanced and set the following e Paper Size 30256 e Halftoning Super Cell 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 Qn It is best to confirm that the shipping label selection has been recognized by checking the set up from within the operating software Open the Data Viewer module and select Page Set 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 pup 19 2
109. velengths are 300 nm for 41 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 normalize the data and spectra using the absorbance value of the wavelength between 400 nm and 700 with the lowest andabsorbance value e Microarray The default setting is SSDNA 33 for the nucleic acid The default settings remain Dye 1 set to Cy3 Dye 2 set to Cy5 with absorbance normalized to the absorbance value at 750nm Other options include RNA 40 ssDNA 33 and Other There are several hard coded dye choices including common Alexa fluor dyes See the explanation of the Dye Chromophore Editor later in this section for information about adding custom dyes and chromophores e A280 There are six sample type options available for purified protein analysis and concentration measurement The default setting is Other protein E196 See Section 8 for additional information about each sample type option Note Software versions 3 5 1 and higher include the option 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 type options available for purified protein analysis and concentration measurement The default setting is Other protein E1946 The user may also elect whether or not to use a bichromatic normalization of the 280 nm absorbance value to the absorbance value at 340 nm The default d
110. y 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 0 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 10 1 Section 10 9BProtein BCA Making BCA Measurements A standard curve is required every time the BCA assay is run Although curves can be saved and reloaded in the NanoDrop 1000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay
111. ye setting 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 calibration check utility is available in software versions 3 5 1 and higher For more information on using this module refer to both Section 15 Calibration Check and Section 16 Troubleshooting of this manual 3 3 Section 3 General Operation 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 deleted User Access Manager All Linens Level Active Locked Expired Exnires

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