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NanoDrop 8000 Spectrophotometer V2.2 User Manual

1. Account Types There are three types of user accounts 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 below Note The administrator or the last level 10 user account may not be deleted 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 in to the user s account in c ND 8000 Data and the user specified location if that preference is selected 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 into the Default folder within the c WD 8000 Data folder Note For laboratories requiring that every user have a unique user account the administrator may disable the default user account
2. 1 2 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 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 may be locked if the incorrect password entry occurs as described above Change Password This module enables each user having an authorized account ID to change their respective password Note The administrator using the Options or the Modify User entr
3. 750 Report 495 A Wavelengths 400 750 Edit Report Wavelengths xi I Use this screen to select the wavelengths of interest as well as the minimum and maximum wavelengths to display on the screen Up to four wavelengths may be included as Report Wavelengths The third window is used to define the baseline correction type 6 2 Section 6 User Methods Edit method parameters Step 3 of 5 Baseline Baseline Correction Type Slope Alinearbaseline correction from ae Baseline Correction Wavelegth 1 to Baseline Correction Baseline Correction Wavelength 1 nm 400 Wavelength 2 is substracted from the raw absorbance spectrum Baseline Correction Wavelength 2 nm 750 Cancel Step 4 allows the option of including results of up to 2 user defined formulas in the display and archived data The formulas are not used in the calculation of the sample concentration Edit method parameters step 4 of 5 Formula You may have up to 2 user defined formulas calculated for this method Select one of the previously defined formulas from Available Formulas or first select Edit List to define a new formula Available Formulas Formulas used with this Method 2 maximum 260 280 Name Formula 260 230 gt gt gt 495 280 A 495 A 260 lt lt lt Edit Formulas Cancel The final page of the wizard allows the user to name and describe the new method Edit m
4. concentration and purity of purified protein MicroArray dye incorporation concentration and purity of nucleic acid UV Vis general UV Vis measurements Cell Cultures absorbance light scattering measurement of suspended microbial cells Proteins amp Labels concentration of dye labeled proteins conjugates and metalloproteins Protein BCA protein concentration using the BCA assay Protein Bradford protein concentration using the Bradford assay Protein Lowry protein concentration using the Modified Lowry assay Protein Pierce 660 nm protein concentration using the new 660 nm assay Note Colorimetric applications are not available in the single sample mode and the associated menu buttons will be grayed out 9 1 Section 5 Standard Methods Nucleic Acids Nucleic acid samples can be readily checked for concentration and quality using the NanoDrop 8000 Spectrophotometer To measure nucleic acid samples select the Nucleic Acid application module on the Main Menu 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 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 Use an 8 channel pipettor
5. Min Max Baseline Units Std Ext Factor Mol h Mode nm nm mm Cre Coef _ Weight_ Caffene Standards 272 220 350 350 mal Linear LDH cell count Standards 490 230 750 750 cellsful Linear T T k 4 La z ias gt Note protected methods are indicated with a diamond and can only be modified by their creator Note Diamonds indicate predefined methods which cannot be modified Create New Method A wizard style series of new windows will appear that will guide the user through the creation of a new method The first window is entitled Measurement Type and is used to select the method of calculating the sample concentration The options allow the user to choose between using Beers Law standard or modified to utilize constants instead of extinction coefficient or standard curves as the means of calculating concentration The Oligo Required option will use the calculated extinction coefficient of the entered oligo sequence in calculating concentration 6 1 Section 6 User Methods A fifth option of none enables the creation of methods that report absorbances at selected wavelengths without calculating concentrations A series of secondary drop down options and required parameter boxes are displayed as appropriate based upon the Quantification Mode selected Note Methods requiring standard curves are not available when operating under
6. are measured Polynomial curve fitting requires more standard points depending on the polynomial degree selected 5 23 Section 5 Standard Methods File Standarda Help Frim Fage Onteut 370 5004 215 PM Anta to Sample Agguisiign 20000 01 8 0I Lewin imitim Measurements Tak Double Click on any w tt change the concesiraton or daleie mplicalas mint Aura Ab Aba 1 Aba 7 Abe Abe 4 Aha 5 _Monsura aos 0004 000 2002 0005 0004 0004 ares 0087 008 0020 006 088 4 mi uias 0045 ga 120049 Gord cies 0085 0083 4107 0107 100 0700 b 000 DUE TE 0212 4 43716 Sterdard Cure Absorbance Spucte ramtmmoog l i i i L L 7 4 400 425 450 475 500 525 550 575 600 G25 650 675 700 7 Wennlcngih e Measure Samples Once a minimum standard curve has been established the standard curve indicator will change from gray invalid to green valid allowing the user to start measuring samples The designation of Valid only indicates that the minimum requirement for a 2 point curve has been met Sample concentrations are 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 Bio Sandoris Hap Purd Page Litar Cretan Ana200s 213 re Fetim to Sample Ace 200001 Tur
7. pm mimi r ms Sample ID ASA a es 1 096 410 mm Hormalizabon Le ERE A E ul 7 j Ara Hi 1 h Ed Gapet j LIE ES i7 O A saple 1 rev abe 1 187 2020 074 Al J Sample ID BSA A GN Ago i 1 762 1 1 12 dei O a 1 Wy Fl sampler 1 mias 1236 seven 074 mail 1 Sample ID ASA Ma E Azan 1 243 1 064 Active 1 F GI fmni al 11 17 L A napis 1 nm Tbe 1 166 2504200 0 75 majral Gare 1D DSA ra A a0 1 159 1 784 Anin aly HP 5 x oe U N Samples H 1 miah 12 w0 O75 afi Sample 1D RSA uk 2280 170 1 936 A al II e 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 Note Concentrations for all eight samples will be calculated using the same mass extinction coefficient as determined by the Sample Type selection A description of each sample type is given below 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 E Bovine Serum Albumin reference Unknown sample protein BSA x concentrations are calculated using the mass extinction coeffic
8. Diagnostics Calibration Check Main Menu 2 7 A CF 8 kit is required to run the calibration check procedure The kit includes 8 well PCR strip tubes and two ampoules of an aqueous potassium dichromate K2Cr2O07 solution CF 1 for use in confirming calibration of NanoDrop 8000 Spectrophotometers The Thermo Scientific NanoDrop 8000 Calibration Check diagnostic utilizes five measurements on each of the 8 positions to verify the pathlength calibration It is important to pay careful attention to technique when performing this procedure as a single outlier caused by user error can result ina failed calibration check on one or more pedestals Required Materials CF 8 Calibration Kit 200 ul Single Channel Pipettor amp tips PR 1 Reconditioning Kit 8 well PCR strips 2 Manual 0 5 to 10 ul 8 channel pipettor Low lint lab wipes 0 1 10 ul low retention pipette tips dH 0 Cleaning Instructions It is strongly recommended that the pedestals are clean as described below before beginning the calibration check procedure 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 Wait 30 seconds for the PR 1 to dry 3 Fold a clean dry laboratory wipe into quarters and remove the PR 1 by aggressively rubbing the surface of the upper and lower measurement surfaces until all the bla
9. For the NanoDrop 8000 to operate successfully a total of three USB devices need to install although only two cycles through the hardware wizard will be observed To confirm installation view the Windows Device Manager as shown below 2 1 Section 2 Initial Set up m Computer Management Local BL System Tools il a j HO Bat El Event Viewer 43 Biometric pa Shared Folders ig Computer ME Local Users and Groups ge Disk drives 9 Performance Logs and Alerts amp Display adapters El Device Manager 12 DVD CD ROM drives 5 Storage ig Human Interface Devices Removable Storage IDE ATA ATAPI controllers Disk Defragmenter 5 IEEE 1394 Bus host controllers pe Disk Management Keyboards i Services and Applications Mice and other pointing devices Ma Modems E Monitors NanoDrop Devices LED Position Indicator ND 8000 Peripheral Control Device ND 8000 Spectrophotometer _ BB Network adapters The NanoDrop 8000 Spectrophotometer should now be ready for operation If the software does not start properly refer to the Troubleshooting section for possible 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 Display Properties by right clicking on the desktop and select Properties gt Appearance Additional step
10. Plenel veel Cancal To move to the next well in a column enter the sample name and then click on the Next Well button To select a well in another column highlight the well of interest enter in the sample name and hit the keyboard Enter button Cancel Closes the window without making any changes 4 3 Section 4 Sample ID Entry Options Note If a Sample ID File format has been defined see below that format will be retained and applied to subsequent plates until the user selects a different format For this reason when loading a predefined list of sample names i e a plate file it is crucial for a user to know whether the sample names should fill in the screen plate map by row sets of 12 or by column sets of 8 Configuration Options for 8 Sample Mode If the user cancels the Plate Setup Mode without loading a plate file the above operations may also be accessed from the Configuration drop down on the main acquisition page In addition the Configuration drop down includes the following options Edit Moda Help Define Sample ID File Format Sure Load Sample ID File Manual Sample ID Entry sample ID Required Auto Advance Columns Prompt Close Data Viewer Measurement Limits show Plate Summary e Sample ID Required If selected the sample ID field must be populated for each sample tested Wells requiring a sample ID will appear red in the sample status color code see below for desc
11. 1 abs current value of the user selectable wavelength cursor and corresponding absorbance value for a 1 mm pathlength The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e ug mL the concentration of the sample The calculated concentration is displayed in the units selected via the units drop down box e Show Report formatted for 200 samples although the buffer size can be modified Bradford Assay Sample Preparation Follow the manufacturer s protocol for the assay including recommended incubation times and temperature Allowing the reaction to incubate for a longer than the suggested time frame increases the potential for interfering aggregates Higher protein concentration may increase the possibility of dye or dye protein aggregates contributing to interfering light scattering In addition to the kit reagents protein standards BSA for generating a standard curve for the Bradford method are often provided by the manufacturer Use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the NanoDrop 8000 can measure higher protein concentrations than a cuvette based spectrophotometer you may need to supply your own protein standards at higher concentrations than routinely provided by the manufacturer Variation Between Different Kits When comparing Coomass
12. Data Export feature by enabling the On box Select 7 1 the data export destination using the icon next to the Data Export Folder dialog window Save the alternative path by clicking on the Save and Exit 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 8000 Spectrophotometer does not need to be connected to the PC to use the Data Viewer module Data Viewer Features The Data Viewer is composed of two or three pages in a tabular form consisting of Plots Reports and Standard Curves where utilized The user may access any page by clicking on the tabs The software opens to the Report page whether accessed through the Main Menu or Show Report Note Recording rather than Start Report must be selected in order to access the Data Viewer via Show Report Tool Bar Features common to all three pages
13. Laai jargia H 1 ASSO O20 Ad 00142 mani 5 She mimi agag 00 CISA O 174 Section 5 Standard Methods e A 650nm the Cu complex s absorbance at 650 nm for the 1 mm pathlength e nm1andnm1 abs current value of the user selectable wavelength cursor and corresponding absorbance value fora 1 mm pathlength The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e mg mL the concentration of the sample The calculated concentration is displayed in the units selected via the units drop down box e Show Report formatted for 200 samples although the buffer size can be modified Modified Lowry Assay Sample Preparation Follow the manufacturer s protocol for the assay including recommended incubation times and temperature In addition to the kit reagents protein standards BSA for generating a standard curve for the Modified Lowry method are often provided by the manufacturer Use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the NanoDrop 8000 can measure higher protein concentrations than a cuvette based spectrophotometer you may need to supply your own protein standards at higher concentrations than routinely provided by the manufacturer Making Lowry Measurements A standard curve is required every time the Modified Lowry as
14. S A Telephone 302 479 7707 Toll Free 1 8770724 7690 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 10 2010 Table of Contents T OVC RV IC Wiis A ad 1 1 Instrument DESCIPlON 1 1 OBEI 0 eenn ee ne nn eve eters Oren ere ee ree te enn ene 1 1 A ediatateelsasoaceenetiae 1 1 WEEE COMDIANGO seis ececets aa 1 2 A eri rset eet eee ere Mer eh eter enced es a oer era 1 2 nta Set Unas 2 1 Computer Requirements 2 1 Software Installaatio Mail eauspedy Meanictvheubenteadecasndatesede 2 1 Registering Your InstruMeNt ococcccnnncoconnncocononoconcnnoconnnconanonononcnconananonos 2 3 3 Gen ral Operator 3 1 Cleaning the Sample Retention SySteM ccococccccccnccccccconncnnnnccnnnnanonnnns 3 1 Sample Size Requirements sessa E 3 2 Sample HOMOGENEILY asear e eaa E e E 3 2 Single vs 8 Sample Modes ooccccccccccccconccnnncccnnconnccnnnncnnnnannnnnnnononnnanennnnnos 3 3 Functions Common to Single and 8 Sample Modes ocooooccccnccccccno 3 3 Functions Specific to the Singl
15. and restart This error message can occur when the software calculates high integration times for particular positions during initialization This is most likely due to the presence of an air bubble or a dried sample left on the measurement surface Cleaning both the top and bottom pedestals with de ionized water and exiting out of the software module to the main menu should alleviate the problem It is not necessary to close the software completely as each module is re initialized when it is opened If the error persists contact your local distributor or Technical Support for assistance Unusual Spectrum A sample that exhibits jagged cuts out of the spectrum but an otherwise normal shape may be the result of detector saturation This can be caused by the software selecting too high of an integration time due to a dirty sample pedestal upon startup Try cleaning lower and upper sample pedestals thoroughly and restarting the software For reference examples of spectra generated with a saturated detector are shown below 8 Section 8 Troubleshootin 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 Sample Accuracy and Reproducibility If you are obtaini
16. appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 21 Unique Screen Features T Lowry File Edit Configuration Standards Help TH Oo AS Measure Blank Retienk Aecoring ShowRepor User Diedeuls Plate M1 Liter slenderds Vir poste Slandards 2 11 200000000000 ele Se 6 6 616 6 6 MO C0OOOCDODOCO 110770 0507 AB Active Oro Actve MH to 1 Sample ID Lewy Date Time 900 2006 2 19 Pt Exit Moassiacrmani corngabelt mm pbi A2 saget 2 AED 0155 ram aber mam mg ml 2 629 Adma m to i Semple 10 Lera Aawa m of 1 Gamp D Lewy Adwa m t 1 Sangis ID Lewy bz Sane 2 Anl 0i mg ml 2 467 C2 Saget 1 4850 03 De Senet 1 AERO ozi Actes Mi El 1 Simple ID Lowey FE Samet 1 ALSO 0216 Active m 1 Sample ID Lowey 4000000000000 T Telo olo olo o lololo S COCO0C0O000 CJ i Active im e 1 Samil 1D Lagar F2 Sangeet 1 Ap Osi Fully 0118 mami E mgm 4 65 mgm 558 24405 0115 mgin a573 G2 Saneti 1 Ato Dir 2405 0110 Active E t Sample L
17. at the time of measurement Note This page is only available for software modules utilizing a Standard Curve File Configuration Data Reports Help Plots Report Standards Test type Bradford 10 27 2005 1 17 Standards Sample Curve Ref Ref Std 1 Std 1 Std2 Std2 Std 3 Std 3 Std 4 ID Type conc Abs conc Abs conc Abs conc Abs conc Reference _ Interp 0 00 0029 NaN NaN NaN NaN NaN NaN NaN Standard 1 interp 000 0 029 100 00 0 047 NaN NaN NaN NaN NaN standard 2 Interp 0 00 0 029 100 00 0 047 1000 00 0 099 NaN NaN NaN Standard 3 Interp 0 00 0 029 100 00 0 047 1000 00 0 108 2000 00 0 073 NaN 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 To open these reports go to the C ND 8000 Data Reports folder and right click on the file of interest Note Save and rename files before making any changes if opened with Excel type of programs to ensure that the original archived data is available for importing using the Data Viewer function Diagnostics and Utilities Calibration Check The Calibration Check is found within the Utilities and Diagnostics module and is accessed through the Main Menu It is used to confirm that both pathlengths are within calibration specifications Utilities amp Diagnos Select Diagnostics or Calibration Check
18. be loaded using the Standards menu bar drop down options 9 27 e Measure Standards Section 5 Standard Methods Up to 5 replicates of each standard can be measured The software will not allow measurement of samples until a minimum of either a reference and 1 standard or 2 standards are measured Polynomial curve fitting requires more standard points depending on the polynomial degree selected Pradlord Handard Curves Filo Stendeeds Hop Prin Page CAOS TONT AM Hetan lo Sample Acguisiban 1000100055210 Heasuremerts Table Sander l Messura y Pitank CHA D Ch B Choe r 5D 145 Eh E Eh F A Ch Eh H e Measure Samples pd lick an any rosy dos menge T fhe concesiratan ar delete replic entes gr 1 wt Ab 20000 0144 40000 0173 ADO E I 205 L i p75 Wiewelength Once a minimum standard curve has been established the standard curve indicator will change from gray invalid to green valid allowing the user to start measuring samples The designation of Valid only indicates that the minimum requirement for a 2 point curve has been met Sample concentrations are 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 Bradford Shende Garnes Ble Standa
19. before exiting the BCA software module 9 20 Section 5 Standard Methods 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 that a standard curve be generated before unknown protein concentrations can be determined The Modified Lowry procedure involves reaction of protein with cupric sulfate in an alkaline solution resulting in the 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 the respective manufacturer s recommendations for all standards and samples unknowns Sample Volume Requirements The presence of surfactants or detergents in reagents can significantly alter the surface tension resulting in difficulty forming and or maintaining adequate columns for measurement The column formation issue 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to dela
20. button Automatic Data Export Y On Export Data By Row Y On Test Module Data Export Folder Nucleic Acid C Documents and Settings dave lach MicroArray Desktop UV Vis Protein A 280 Cell Culture Proteins amp Labels BCA Protein Configure Data Bradford L Export for Module owry Pierce 660nm Custom cancel Selecting the Configure Data Export for Module button will bring up the following pop up box 7 4 Nucleic Acid Data Export Configuration Select the data expor fields and the order thatthe fields should appear Change the order by selecting and draqging an item to the desired position Optinally enable the export of the raw absorbance data range and log interval Export Fields Abs Spectrum Export Plate ID A Well show All Sample ID Disabled _ User ID Date Move Up Amin 220 2 Time ngul en Move Down Amax 350 A260 260 280 Delta A 1 260 230 Delete Constant Cursor Pos Cursor abs This box is used to select the particular data columns of interest when exporting data Save the designated path by clicking on the Save amp Exit button before exiting the User Preferences module Note The user may select specific default report configurations for each pre defined method As seen in the image below only one default report formula is available for all user defined methods Use the drop down Load Report Format to utilize a different saved configuration wh
21. configuration drop down menu minimum and maximum concentration limits can be set for protein measurements as defined by the absorbance at 280 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 9 Section 5 Standard Methods Unique Screen Features T Protein A 50 E Elis Edhi 3 Conhepurahen lanar Hein AAA o A TT a aE Measure Blank Reblank Recording Show Repon Le Default Deta Time 9 10 2000 9454M Ent Pisto IL ABA Mrasurmirmaal corral A sample Type OSA wi All Active Gini nm 200 2 Unis mgin Ae t 1 P _Al Sample H 1 Tabs 1 130 A A alv gimi Gapel ESA an E A280 1 156 1 733 Ariva Yj 1 A Bl Ss 12 7 LI A Sample Ht 1 ren Tab 1206 mam O75 agimi Sample ID ISA 1 AS 1 214 1 521 Ache a 1 AEI ener a O A ample 1 mmia 1 181 awm a mg ml Sample ID ISA iE O Azil 1 166 1 779 Arin 4 4 DI Sample 1 Taba 1258 PLAN 074 A
22. covered by sample Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Measurement Concentration Range The NanoDrop 8000 Spectrophotometer is configured to measure absorbance up to the 1 mm pathlength equivalent of 7 5 A when utilizing the 0 2 mm path via the automatic path selection feature By selecting Measurement Limits from the configuration drop down menu minimum and maximum absorbance limits can be set for the shorter of the two user selectable wavelengths These limits cannot be set as a default and must be defined each time the application module is opened Sample absorbances that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The absorbances will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 12 Section 5 Standard Methods Unique Screen Features AS Bile Edil Conhqurabon ini Help y Measure Diank Rerblank Roconi Show Repon Liso Default DateTime 32000 2 20 Phil Exit Pisin II Oichomeatie Anomatc Path Selecson l On Measu
23. dirty make sure surfaces are clean Sample arm make sure sample arm is in down position No power to instrument check 12 power supply For more details 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 the problem perform an Intensity Check e Open the Utilities and Diagnostics module from the main menu e With the sampling arm down select OK to initialize the spectrometer and then select Intensity Check You will see two panels of 8 spectra each and a bias value greater than 65 as shown below This indicates that the USB communication is normal the power supply is operational and the flashlamp is functioning Tarini humor USDBAGIZTISCPEI Pedostal Hi a Dsiecior fies FH Ee ie n a 4 r B L 1 4 4 i 1 1 t S30 Tb Apa Spa 060 7500 300 3000 2000 nii Bao 106 Woo 260 Mtra rg am Wigan Aone ite Single Dii 0 54 non Peskig Mos orad ilam 446 Paro oe S60 Tim 2805 le Mibnm Wii f232nm 8792 I U f 1 i 1 i i p p 1 1 200 2000 2340 4060 4500 500 0 BE BO 100 7509 5600 2500 4 m ray say e 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 vol
24. down includes the following two options e Sample ID Required If selected the sample ID field must be populated prior to each measurement e Prompt Close Data Viewer If selected the user will be given the option of closing the Data Viewer when exiting the software module 4 1 Section 4 Sample ID Entry Options Sample ID entry 8 Sample Mode Selecting the Sample Loading Mode After the instrument has completed the initialization process the following window will automatically display the options for plate setup Select Sample Loading Mode Load Sample ID Manual Sample ID File Entry Define Sample ID Cancel File Format Load Sample ID File The NanoDrop 8000 offers several options for entering sample IDs When making only a few measurements it is easy to simply type in sample names prior to measurement or use the Manual Sample ID Entry The NanoDrop 8000 software also enables the user to load a list of predefined sample IDs or names which improves efficiency and reduces errors when measuring many samples The Load Sample ID File option opens the Plate file folder enabling the user to select a list of pre defined sample IDs The lists may be created in Excel or Notepad but all lists must be saved as a txt file It is recommended that the files be stored in the Plate Files folder at C IND 8000 Data When creating a file enter all sample names in one column and the number of replicates desired for each sample in anot
25. focused meaning it is the default selection on the screen and should have a flashing text cursor when the instrument is waiting to make a new measurement See the section on Sample ID List plate Format for additional information regarding the entry of sample IDs 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 3 5 Section 3 General Operation Functions Specific to the Single Sample Mode T Aochtie Acid Simple Sajah ji File Edit Configuration Tindi Help o __ Mensura Blank Pie blank Recording ShowReport User Default Dete Time 3192009 402 PM Ea Measurement complete M Sampla DO DMNA sample 6 Measurement Results Units noul ha Asoy 049 nm 1 abs 1448 6080 1 86 Semplo Typo B A760 1448 2501230 200 ngul Bana nm 1 260 Chera control Citar graph now s Longini 11 28 DMA samples ys DMA sample 5 se DMA sampled ws DMA sample 3 re DMA sample e a OMA sample s55 4Ansobarce mm 114 s i i i i i i i i i i i i i 220 230 240 250 260 270 280 90 300 310 320 330 340 350 Wesnwelengih nm 27 0 CA AAA Load Next Sample ID F7 This button enables the user to automatically load the
26. for the nucleic acid component as defined by the absorbance at 260 nm These limits cannot be set as a default and must be defined each time the application module is opened Nucleic acid measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 9 Section 5 Standard Methods Unique Screen Features T MicroArray E Pd Ebor Eeh Conhepurahon moderado Hop zE o a a ee ee IMM Measure Blank Rebienk Recording Show Report User Default Date Time 9 9 2000 215 PH Ei A y x 4 A A A A A A Pisto IL Measurtmmand cripte sl Sample Type 500H4 13 se All Active Onin nm 260 E Lim mogul W Acre m t 1 AR comple H 1 Dye liebe 0351 Dye Tobal 23 40 ngful Sample ID pays SS Tabs 015 Dye Fabs DES Dyn pmol 2553 33 19 Acton m H 1 oF Sample H 1 Dyr 1 ata 01351 Dye 1 pmol 23437 nqful Samples ID Opa KO Aah rm sbe 0117 Dee tebe UES Dye E peobul 25 50 34 AR Actors Mi ft 1 ER Semple 1 Dye late 0S2 Dye lpenolul 234 Dre 1 Cyd ai A
27. id adds 6 1 Edit Selected Metho0d cccccccocccnccccnncccnononcconnncnonononcnonnnonnnonnnnnnnnononanenons 6 4 VIS A o dd o 6 4 User Method Acquisition Screen ooonnnccnnccccccnonccnnnccnononnnnnnnnonononanenons 6 5 T TOOIS amp Confi rati n ui da 7 1 SN tee eevee Pr a ee ete 7 1 VEN MCW CN I batacpetite da aehas teat E te aetend Ana ee bentaaauesedusasieeusenaee 7 2 MPOr rage erent gee eee eee nee eee ee eee 7 2 A A eek aca lenigane eal aes 7 4 MEDOM PAQC rr tr tibio 7 4 standards P agarren mas adarnedn emcebedesauemeasonamiecosascude 7 7 Diagnostics and Utilities ccoooconcnncccconcnnnnnnonnnnnononnnnnnononnnonnonanonns 7 7 Gallbration Checa at ee a aaa aide ert 7 7 A a N 7 1 Account Management lll 7 2 User Preference Sorteo ii 7 3 8 Troubleshoo IN Sii 8 1 ETFO OCC Saori cra 8 1 Unrecognized Devices cccccooonocccnncccccnnoocononcnononannconnnnnnnnnaancnnnnnnnonanennos 8 1 Connection Erica gi 8 3 A R ee dad E 8 4 Hs A 8 5 Eror Code o ano pieiod 8 5 Liquid Column Breakage ooocccccccccncccccccccooononnnnnnnnnnnnnnnnnnnnnonononanannnnnnnos 8 6 Unusual SP CUMIN e ea a a a lle 8 7 Sample Accuracy and Reproducibility ccccccccececeeesssseseeeeeeeeeeeeeeees 8 8 ASA A me eet rae eae ete 8 9 Technical SUP DON rena ocean rocosa ias 8 10 9 Maintenance and Warranty cooonccccnnnncccnnnncncnnoncoconannonennanonrnaannnnnans 9 1 A Ruseuen aan ecu etal eee emia iycicnat aio ances 9 1 D
28. list file before measuring samples e Confirm that reference blank solution and solvent are the same material Buffers often absorb in the UV range and therefore it is critical to blank the instrument with exactly the same material that the sample is Suspended in e 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 table of concentration ranges provided within the respective application module section of the manual for lower detection limits e Confirm instrument accuracy with CF 1 CF 1 is a concentrated potassium dichromate calibration standard and is manufactured exclusively for use with NanoDrop instruments and available from Thermo Fisher Scientific and its distributors It is a good practice to check the instrument s performance every six months with fresh CF 1 260 280 Ratio Many researchers encounter a consistent 260 280 ratio change when switching from a standard cuvette spectrophotometer to the NanoDrop 8000 Spectrophotometer The three main causes for this are listed below e Change in sample acidity M 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 8000 Spectrophotometer to other spectrophotometers it is important to ensure that the pH and ion
29. next sample ID when utilizing an imported list txt file of sample names Note This button will only be displayed when a list of sample IDs has been loaded Legend The most recent sample ID will be listed under the Legend heading on the right side of the screen When the Accumulate until clear overlay control is selected the most recent sample measured will be displayed at the top of the list Note A maximum of 12 sample IDs will be displayed in the list at any one time When more than 12 samples are measured without clearing the spectrum the earliest sample IDs will be removed from the legend list Note The Single Sample acquisition screen for each module is similar to the one displayed above Screen shots of the single sample mode will not be presented for each application in section five 3 6 Section 3 General Operation Functions Specific to the 8 Sample Mode Eta Eai Coonhcgureaticn Hop Measure Diank Rebiak Recording ShowRepor User Default DmsTime 992000 221PM Ei A A 2 td Pista IL 425 ngil Measurtmmand comripleste E E sample Type o DA lw All Active OOH fd 260 Wines mel Actors m t 1 ea 5h AJ Semple 7 rm laos EET Aga Em T Sampie D 440 rpd i Awm amg mwan 12 a 219 ea Acton fm H is Ae HI Sample 1 mmia 88E AOR RASE ali Sample 10 dl nt Aa AGS 35030 1 65 A 219 pica Actors Mi ft 1 CI Sample a 1 re
30. not select an application or method folder within a user in this activity box e 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 e Select or Deselect Used to move the highlighted sample choices to or from the Selected Samples box Note The software defaults to a buffer size of 1000 samples e Search Function allows the user to locate specific data by searching through sample ID names e Sample Information and Spectrum Are populated with the information associated with the most recently highlighted sample e 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 7 3 Section 7 Tools amp Configuration Plots Page The Plots page displays selected sample spectra Features include e Test Type Auto fills in module name e Date Auto fills in date and time of report e 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 Sel
31. the Single Sample mode The following screen shots illustrate the steps involved with setting up a custom method using a potential Alexa 488 dye method as an example Edit method parameters Step 1 of 5 Measurement Type The concentration is calculated by dividing the absorbance A at the Analysis wavelength by Chromophore Alexa Fluor 488 v Extinction Coefficient times the path length Both the Analysis _ nm and the Extinction Coefficient Extinction coeff ifmolcm 7 100E 4 are required parameters The concentration will be returned in the selected Units For weight based units a Molecular Weight is required Quantification Mode c A bxExt Coeff v Analysis Wavelength nm 495 Units ma v Mol Weight g mol 0 000E 0 Cancel Selecting the Next button will bring up the Wavelengths screen Edit method parameters Step 2 of 5 Wavelengths The absorbance at all wavelengths between the Plot Min Wavelength and Plot Max Wavelengths are automatically archived for each measurement Use the Report Wavelengths listto define wavelengths of particular interest for tabular absorbance display during acquisitian and automatic inclusion in reports The Wavelengths listis pre populated with the Analysis and Baseline Correction Wavelengths Click on Edit Wavelengths to change wavelengths or define additional wavelengths Minimum Wavelength nm E 0 Maximum Wavelength nm
32. want to uninstall the Thermo Scientific NanoDrop Product and keep the NanoDrop 2000 If there are two instances of a NanoDrop 2000 device uninstall one and keep the other If problems continue install the instrument on another PC to rule out a faulty USB hub port on the original PC If the instrument is still not recognized then the instrument may need service Contact your local distributor or Technical Support for assistance 8 2 Connection Error Connection Error There was an error communicating with the instrument Try the following Check the USB cable and select Retry If Retry fails disconnect the USB cable and then reconnect and select Retry again this does not solve the problem refer to the Troubleshooting section of the User s Manual available from Main Menu This error occurs whenever the USB connection is disrupted while operating a software module Ensure that the USB cable is firmly inserted into the instrument and the computer then select Retry In most cases this will correct the problem When attaching the USB cable please wait at least 30 seconds for the USB devices and internal drivers to be installed and recognized Some additional possible causes for the error message 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 r
33. warning persists and the above remedies do not solve the problem contact NanoDrop Technologies or your distributor OPEN USERS MANUAL This warning occurs when a possible problem with the column is detected The software compares the long path and short path absorbances and issues a warning to the user if the short path is not 20 of the long path absorbance within a tolerance The most common explanation is that the column is not forming properly due to the pedestal being unconditioned When a pedestal becomes unconditioned sample droplets applied to the bottom pedestal will flatten out and cover the entire pedestal surface rather than 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 result in difficulty forming columns with 1 ul samples Pedestal 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 8 6 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
34. 0 0 616 616 66 FR SOC OOOO OOOOU TIG67750 AMY 200 a e A 562nm the Cu BCA complex s absorbance at 562 nm for the 1 mm pathlength e nm1andnm1 abs current value of the user selectable wavelength cursor and corresponding absorbance value fora 1 mm pathlength The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e mg mL the concentration of the sample unknown The calculated concentration is displayed in the units selected via the units drop down box e Show Report formatted for 200 samples although the buffer size can be modified BCA Assay Sample Preparation Follow the manufacturer s protocol for the assay including recommended incubation times and temperature In addition to the kit reagents protein standards BSA for generating a standard curve for the BCA method are often provided by the manufacturer Use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the NanoDrop 8000 can measure higher protein concentrations than a cuvette based spectrophotometer you may need to supply your own protein standards at higher concentrations than routinely provided by the manufacturer 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 8
35. 0 0n 0 Iw Active 3 e A660 nm protein dye complex s absorbance at 660 nm at the 1mm pathlength e nm1andnm1 abs current value of the user selectable wavelength cursor and corresponding absorbance value fora 1 mm pathlength The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e ug mL the concentration of the sample unknown e Show Report formatted for 200 samples although the buffer size can be modified Making Pierce 660 nm Protein Measurements A standard curve is required by the software every time the Pierce Protein 660 nm assay is run Although curves can be saved and reloaded in the NanoDrop 8000 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 meas
36. 00 ug ml up to 8000 ug ml on the NanoDrop 8000 The best linearity is in the 100ug ml 1000 ug ml range The concentration range for the mini Bradford assay is 15 ug ml to 125 ug ml By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be set for protein mg ml measured with this method at 595 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing Coomassie dye dye and Coomassie dye protein aggregates are frequently encountered in Coomassie dye based protein assays With time particulate can be observed which can cause significant fluctuations in Absorbance readings It is also important to note the total analyte protein dye signal at 595nm is limited to 0 150 A as a result of the 1 0mm pathlength of the instrument the Bradford Coomassie dye reagent concentration and the acidic pH Making measurements in triplicate of standards and samples unknowns is good practice particularly with the limited assay signal obtained with the Bradford Assay 9 25 Secti
37. 000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay Both single and multi point standard curve generation are 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 displays a maximum of 5 replicates for each of 7 different standards There is no set order in which standards must be run The following box will appear after the module initialization is complete 5 18 Section 5 Standard Methods Choose Standards Source Standards are required Choose the Standard Curve source Load Standard New Standards Curve Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Measurement Table Double ick n Arry now do changa h concentration or dels ricas Do you wantto load the Ache E A Felerance 0 000 concentrations and curve type for Active WI 8 Stendori1 1000 yp Active E C Stenderd 2000 O 3 000 E Standard 4 F Standard 5 generating a standard curve from a previously saved standard curve file fl Starland 7 Clicking on the Yes button will allow the user to import just the Standard series without the respective measured value
38. 1 rmi 000 Ade Dae aS ego A Te CHL 187 Aa 213 y 000000000 0000734 0470 e 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 Oligo Calc and Other for other nucleic acids The default is DNA 50 If Other is selected the user can select an analysis constant between 15 150 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 Choosing the Oligo Calc option will allow the user to enter a defined oligo sequence The molecular weight molar extinction coefficient and the concentration factor specific to that sequence will be displayed Sequences can be cut and pasted into the box from other sources Use the browse button on the left of the Oligo box on the acquisition page to modify the sequence See the Section below Unique Screen features for more details regarding the use of this feature as a stand alone Oligo Calculator e nm 1 and nm 1 abs current value of the user selectable wavelength cursor and corresponding 10 mm equivalent normalized absorbance at the respective wavelength absorbance The wavelength can be set by using the up dow
39. 601 4602 4595 4611 461 3 4598 460 0 4570 4595 4571 4585 4583 4586 4642 4593 4605 4632 4597 4596 4575 4585 4565 EN 4568 456 0 4593 4575 4583 4586 4595 4596 4614 4622 4603 4616 4598 4586 4648 461 6 4623 461 3 461 7 4608 H 4586 4599 4569 4586 4574 4590 4608 4587 4585 4605 4606 4602 Cell Color Code White inside limits Red outside limits Light green inside limits amp selected Repeat Selected Clase Window Dark green outside limits amp selected Click on any cell to select de select that cell for repeat measurements 4 6 Section 5 Standard Methods 5 Standard Methods Software Architecture and Features Main Menu With the sampling arm in the down position start the operating software by selecting the following path Start gt Programs gt NanoDrop gt ND 8000 version The software opens to display three tabs including Standard Methods User Methods and Tools amp Configuration NanoDrop 8000 V2 1 0 File Help User Default v Standard Methods User Methods Tools amp Configuration Single Sample 8 Sample Proteins amp Nucleic Acid Labels Protein Protein A260 Protein MicroArray Bradford Protein UV Vis Lowry Cell Cultures Protein Pierce 660 nm Applications The operating software has been tailored to meet the life scientist s needs It includes the following pre configured application modules Nucleic Acid concentration and purity of nucleic acid Protein A280
40. 8000 Using a 1 1 reagent to sample volume dilution the concentration range of detection is 0 01 mg ml 0 20 mg ml By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be set for protein mg ml measured with this method at 562 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 17 Section 5 Standard Methods Unique Screen Features I BLA Protein File Edit Configuration Standards Help Measure Blank Re blank Puschnig Show Report Liser Dietault Date Time 9 10 2000 11 72 A Plate i BLA Muasiscernenl complete A AT bho oe Standards Las Active Cnt mm 262 i Vir Jpcialo Standards N lida Acie f 1 5 1 nm 7 abe Sample ID BLA ASET AHE as Adma of 1 Sample 1 Sie 618 8 618 619 8 8 SODODOCOCOCO SSODODODODOGON SiO 0
41. Adm El 1 N DI Saree 1 misa Uei Aa We Sample 10 i a Aces 1 N El Sempet 1 mise 01453 ABM uiw 1 EIS ja me SBOODOOOOOO0D0 Acive _ E a FIL Semele 1 nm Tabs 0470 A600 0 084 a gt YO CDOCOCOODODOOO snein _ cCOoO0CODOCODCOCO 5 awel a 4 Gl Sample 1 mmtabe 0467 asi 0 07 Sampir ID F DR J i Ache E 1 Hl Sampat nlab 0471 4600 00 aecoacocacago KE HBO OOOO y 200 CPET GUO e 600nm Absorbance current value of the absorbance at the 11 cursor with the baseline absorbance subtracted Note The actual 1 mm absorbance is displayed e nm 1 current value of the user selectable wavelength cursor and corresponding absorbance The wavelength can be set by using the up down arrows or typing in the desired wavelength e Show Report formatted for 200 samples although the buffer size can be modified 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 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectropho
42. E C Stendend 2000 O 3 000 generating a standard curve from a previously saved standard curve file Clicking on the Yes button will allow the user to import just the Standard series without the respective measured values See image on the right above This option is very useful when running a routine series of standards Selecting No enables the user to enter new concentrations values for standards 1 7 The reference should remain set at 0 00 The Standards menu drop down may also be used to load a previously saved curve generate a new standard curve or view the current standard curve File Edit Configuration ENTERO Help Load from file Measure Blank View or Measure Standards Show Report Then follow the steps below to either generate or modify the curve as needed e Enter the concentration for each standard The user may either click on the Active Inactive box to the left of each standard or double click any where in the row of a particular standard to bring up the Edit Standard dialog box to enter in the concentrations for each standard This box is also used to delete a single absorbance replicate value or reset the entire standard Edit one standard Standard Standard 4 ug ml 1000 0 Average absorbance 0 124 Abs Replicates Delete 0 122 Selected 0 123 Seo 0 129 0 123 Reset Standard 0 125 3 Alternatively previously saved standard curves and standard curve concentration series may
43. EFORE installing this version Current saved User Preferences will be preserved during the uninstall process Note 3 Administrative privileges are required to install this software Note 4 All users of this software must have read and write access to the folder CANANODROP DATA and all of its subfolders When attaching the USB cable please wait at least 30 seconds for the USB devices and internal drivers to be installed and recognized before opening the software To properly install the operating software 1 2 Close all programs and make sure that the USB cable is unplugged Insert the operating software CD in the CD drive of the PC The software installation menu should appear automatically If software menu does not appear choose My Computer to view the contents of the CD Double click on the file named nd 8000 install exe After software installation plug the power supply into the instrument wait 5 seconds then connect the USB cable to the instrument and the computer The Found New Hardware Wizard should start 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 For other Windows operating systems follow the prompts for automatic installation of the software Installation will require two cycles through the Found New Hardware Wizard once for the NanoDrop 8000 Spectrophotometer and once for two devices internal to the instrument
44. Messurembres Table Cute Cid on ny ire lo chapa the concertotion ce delete replicate ree eta toe tae pa Modified Lowry Standard A Curve 0 2 4 0 mg ml la Sree Cursa Abiobante Spica 035 Disda Curve Ty po neral mu e ls A 01i a q i Feigiirei F AT ry 039 M p k k t t F t t t t T i i i i i i t i i i i 00 05 10 15 20 5 30 35 40 45 50 55 60 65 70 75 mai Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module 9 24 Section 5 Standard Methods 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 method the Bradford method r requires that a standard curve be generated before unknown protein concentrations can be determined 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 Sample Volume Re
45. NanoDrop 8000 Spectrophotometer V2 2 User 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 2008 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 19810 U
46. Sample Mode when operating within the Single Sample Mode 2 Carefully withdraw the pipettor before releasing the pipettor s dispensing mechanism Visually verify all samples are correctly transferred to their respective pedestals If some samples have not transferred adequately it is recommended that the aliquots be wiped away with a lab wipe and fresh aliquots of all eight samples be reloaded to ensure consistent loading measurement timing between samples 3 Close the sampling arm and initiate a spectral measurement using the operating software on the PC The sample columns are automatically drawn between the upper and lower measurement pedestals and the spectral measurement made 4 When the measurement is complete open the sampling arm and wipe the samples from both the upper and lower pedestals using a soft laboratory wipe Cleaning the Sample Retention System Wiping the sample from both the upper and lower pedestals upon completion of each sample measurement is usually sufficient to prevent sample carryover and avoid residue buildup Although generally not necessary 2 ul water aliquots can be used to clean the measurement surfaces after particularly high concentration samples to ensure no residual sample is retained on either pedestal After measuring a large number of samples it is recommended that the areas around the upper and lower pedestals be cleaned thoroughly by using a lab wipe dampened with dH20 A final cleaning of a
47. TTCCCGGG Nucleic Acid DNA v Phosphorylated C Double stranded C Mol Weight g mol 3 645E 3 GC 50 00 Ext Coeff Il mol cm 1 280E 5 Conc Factor ng ul 28 48 of bases 12 A Modification Weight oo a Section 5 Standard Methods The calculator allows the user to define the sequence and include relevant information such as whether the oligo is phosphorylated or double stranded The information boxes in the bottom of the screen automatically populated based about the sequence entered Baseline Calculation amp Normalization The software normalizes the visual spectrum display for all readings at 750 nm Normalization for the fluorescent dyes is based upon a software determined slope between 400 and 750 nm for dye concentration calculations Section 5 Standard Methods UV VIS The UV VIS Absorbance module allows the NanoDrop 8000 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 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
48. V sample range 0 15 5 mg ml 0 15 Purified BSA 0 15 mg ml 20 mg ml mg ml sample range gt 10mg ml 2 5 sample range 0 25 4 pmol ul 0 25 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be defined for the protein component in mg ml as defined by the absorbance at 280 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 15 Unique Screen Features T Proteina Labels Section 5 Standard Methods Elo Eei Conheyurahen sterile Help A es ee Oe Measure Blank Re blank Encoding Show Report Lawr Default Date Time yzl 327 PH Exit rr i ee sk a Se 71 eS eee E atid Prato IL TU pastes LIMAT Kieasurtrnen comple ss a E sample Type ESA w All Acre Oni id 200 E Wines mgin Ace 1 Al Semple 3 Dye abe 4 506 Dye ul SOM mg ml Sample Dy BSA gya ah mia 518 Desb MaN Ci
49. aching the USB cable please wait at least 30 seconds for the USB devices and internal drivers to be installed and recognized Report Format Error This error occurs when the user does not have Write access to one of the report format files located at c ND 8000 Data custom report formats or the file has been moved from this folder This error is similar to Error Code 8 see above 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 e 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 e No Active Samples for Measurement This error indicates that there are not any sample positions currently selected for measurement The user may click either All Active or select individual positions for the next measurement e Error 8005 8 5 This error occurs when trying to load a plate file that is not in a txt format e 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 C ND 8000 Data Log Files folder e Error 9003 This error indicates that the monito
50. aggressively rubbing the surface of the upper and lower pedestals until all compound residue is removed Note The black appearance of the removed residue is normal The reconditioning process is complete once the laboratory wipe shows no more black residue To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up As an alternative to using the PR 1 Kit the pedestals may be reconditioned us 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 pedestals and buff rub very aggressively at least 50 times the lab wipe will rip during this procedure and will have to be refolded several times throughout the procedure The upper pedestals may also be buffed but care should be taken not to put too much force on the upper arm To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up If the warning persists and the user visually confirms that the liquid column is forming contact your local distributor or Technical Support for assistance Saturated Detector The detector is saturated for the following channels B This is most likely caused by initialization with dirty measurement pedestals Ifthe error persists clean the pedestals exit the software
51. ames in either the 8 Sample or Single Sample mode The lists may be created in Excel or Notepad but all lists must be saved as a txt file It is recommended that the files be stored in the Plate Files folder at C IND 8000 Data When creating a file enter all sample names in a single file Do not include a column header A barcode reader can be used to scan in individual bar coded sample names into the txt file The column format enables the user to predefine and load an unlimited number of sample IDs using just one file Manual Entry This option opens the Enter Sample IDs window enabling the user to manually enter samples IDs as prompted by the following screen Enter sample IDs x Sampla Cie Enter the Sample Ds vie the koybosrd 5 barcod Of DAPcodd SCANKEGr Alternatively a sample name may be entered directly into the sample ID box on the acquisition screen prior to each measurement Barcode scanners may also be used to enter sample IDs After each entry use either the Add sample ID button or the keyboard Enter key to add the sample to the Sample ID list on the right side of the box Use the scroll feature to view sample IDs entered at the bottom of the list Configuration Options for Single Sample Mode If the user cancels the Plate Setup Mode without loading a list file or manually entering sample IDs the entry operations may be accessed from the Configuration drop down on the main acquisition page In addition the drop
52. ample WO Uger ld Date Tints rol AL AD AA AS Corsia Cursar Pos Cumsor abe 340 rr Messi 6 Ciefault 114008 534 Aid Oo N E 230 O Bilark Ea Default 114062005 6 39 Ah 351 oor 0 03 202 212 0 000 O Measura B Default 1112005 8 29 Ab 43 76 0 80 0 52 1 6 2 43 50 JU 0 6 0 000 MHeasure a Default 1112005 5 40 Ab 203 77 FOF 4 27 1 55 2 5 50 JU 3 1573 0 000 Measure 10 Default 1112005 6 41 AM 3704 14 74 00 39 04 1 06 2 24 50 200 39 075 0 030 Measure Data Storage Hierarchy The hierarchy for archived files is as follows C ND 8000 Data gt User name gt Application Module BCA Protein Lowry Bradford Cell Culture MicroArray Nucleic Acid Protein A 280 Proteins amp Labels UV Vis All archived data files are stored in an application module folder that is within the User folder as shown below Address DB C ND 8000 DatalDefaultiMicroArray Go Folders x Name Size Type Date Modified S ND 8000 Data A S Microarray 2007 03 21 v1 14KB NDS File 3 21 2007 4 02 PM E E Administrator Sl MicroArray 2007 03 23 vl 18KB NDS File 3 23 2007 8 57 AM a Custom report Formats E 2 Default O Cell Culture E 4 Nucleic Acid User Defined Data File Export Location In addition to the primary data storage users may elect to export their data as to an additional location This option can be chosen under the Data Export Exporting tab in User Preferences from within the Tools amp Configuration tab Select the Automatic
53. ance pipet a fresh aliquot before making valios e More than 10 seconds elapsed the measurement between dispensing the samples and clicking the Measure button Tip Practice the Discharge and Touch Sample Delivery until consistent delivery is routine and quick e Evaporation of samples due to e Move instrument to more suitable nearby air vents or exhaust fans location e Position the instrument to best utilize the tip guide e Use a low volume manual 8 channel e Delivery of aliquots not consistent pipettor with tight fitting tips Tip Short rigid tips are recommended for ease of alignment High with the pedestals Standard AO peter Megat e ALWAYS use a 8 chamnel pipettor deviations deliver CF 1 aliquots e Always perform a very quick gt 5 e Insufficient volumes delivered to seconds assessment of the pipette pedestals tips to ensure equal volumes of CF 1 is both drawn up and dispensed single set of aliquots measurement single set of pipette tips measurement Low e Clean the pedestals make a new absorbance e Improper blank measured blank measurement select the values Reset button If the software indicates that recalibration is necessary contact your local distributor or Technical Support for assistance Reviewing Previous Calibration Check Results Previous results may be reviewed at any time by using the Load Cal Check File feature avaialble under the File drop down menu Additional informat
54. 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 to Technical Support e 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 to Technical Support The archived file can be found at C ND 8000 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 8 10 Section 9 Maintenance and Warranty 9 Maintenance and Warranty Cleaning The primary maintenance requirement of the NanoDrop 8000 Spectrophotometer is to keep 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 A final cleaning of all surfaces with de ionized water is also recommended after the user s last measurement Note Do not use a squirt bottle to apply bleach or de ionized water 1 Apply 5 ul of dH20 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 form each upper and lower pedestal with a clean lab wipe Note Typically dH20 is sufficient for removal of samples that have dried on the optical pede
55. and or maintaining adequate columns for measurement The column formation issue 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Measurement Concentration Range The assay has a linear range for BSA of 50 2000 ug ml using a reagent to sample ratio of 15 1 Nees Approx Approx Typical Reproducibility T E Lower Upper minimum 5 replicates yp Limit Limit SD mg ml CV 50 ug ml 2000 ug ml 5 over entire range 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 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 8000 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 By selecting Measurement Limits from the configuration d
56. are referenced off of this zero 5 4 Section 5 Standard Methods Protein A280 Proteins unlike nucleic acids can exhibit considerable diversity The A280 method is applicable to purified proteins exhibiting absorbance at 280 nm 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 in difficulty forming adequate columns for measurement The column formation issue 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measuremen
57. ate a new standard curve view the current standard curve or manually enter in standard curve values Follow the steps below to either generate or modify the curve as needed e Enter the concentration for each standard The user may either click on the Active Inactive box to the left of each standard or double click any where in the row of a particular standard to bring up the Edit Standard dialog box to enter in the concentrations for each standard This box is also used to delete a single absorbance replicate value or reset the entire standard Edit one standard Standard Standard 4 mg ml 750 0 Average absorbance 0 120 Abs Replicates 0 120 A 0 118 0 119 Reset 0 121 Standard 0 123 3 Cancel Delete Selected Alternatively previously saved standard curves and standard curve concentration series may be loaded using the Standards menu bar drop down options e Measure Standards Up to 5 replicates of each standard can be measured The software will not allow measurement of samples until a minimum of either a reference and 1 standard or 2 standards are measured Polynomial curve fitting requires more standard points depending on the polynomial degree selected 9 31 Section 5 Standard Methods Pierce 660nm Standard Curves File Standards Help Default 9 9 2008 4 03 PM Return to Sample Acquisition 2 0 0 00158 0 30 28 Units ug ml Measurements Table Double Click on any row to change the concentrati
58. atibility appendix A final cleaning of all surfaces with de ionized water is also recommended after the user s last measurement Note Do not use a squirt bottle to apply bleach or de ionized water Routine use of ethanol or isopropanol for cleaning is not recommended 9 35 Section 6 User Methods 6 User Methods Users Methods are customized absorbance analysis methods that allow the scientist to create save and edit a set of parameters for both unique and routine measurements All user defined methods except for those requiring standard curves are accessible for both 8 Sample and Single Sample operating modes In the left hand box are the current available user configurable methods Highlighting a method will display whatever descriptive text is associated with the method in the description box to the right NanoDrop 8000 V2 0 File Help User Default v Exit Standard Methods User Methods Tools amp Configuration User Methods Description This is a protected method Oy dye used to determine the concentration based upon absorbance not fluorescence measurements Method Editor Double click to Run Method Method Editor The Method Editor is used to Create Delete Edit and View and Save methods Use the button on the bottom right to access the following screen E gt Sort by Method Name v Method List Method Quant Analysis
59. atio is appreciably lower this may indicate the presence of co purified contaminants e Concentration ng ul sample concentration based on the absorbance at 260 nm and the selected analysis constant The calculated concentration is displayed in the units selected via the units drop down box The units will default to ng ul each time the software is opened See the Concentration Calculation Beer s Law in the appendix for more details on this calculation e Show Report formatted for 200 samples although the buffer size can be modified Oligo Calculator The Oligo Calculator enables the user to type in a specific sequence and choose parameters associated with the oligo When the Oligo Calc sample type option is first selected the following box will appear Oligo Property Calculator x Oligo Seq AAATTTCCCGGG Nucleic Acid DNA w Modification Weight 0 0 s Phosphorylated C Double stranded C Mol Weight g mol 3 645E 3 GC 50 00 Ext Coeff I mol cm 1 280E 5 Conc Factor nguh 28 48 of bases 12 The calculator allows the user to define the sequence and include relevant information such as whether the oligo is phosphorylated or double stranded The information boxes in the bottom of the screen are automatically populated based about the sequence entered 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
60. atory wipe is highly effective in eliminating carryover for samples differing in concentration by as much as three orders of magnitude see our website for NanoDrop 1000 carryover data 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 other highly concentrated nucleic acids are common examples known to the molecular biologist Proteins are subject to denaturation precipitation and aggregation and therefore may require special handling to ensure sample homogeneity Effect of Evaporation and Solvents Evaporation of the sample during the measurement cycle usually has just a minimal effect on absorbance readings and may result in a 1 2 increase in sample concentration This can be observed in the field by measuring the same sample successively over time Highly volatile solvents such as hexane will likely evaporate before the measurement can be completed Less volatile solvents such as DMSO can be used successfully To minimize the effects of evaporation t is recommended that an 8 channel low volume pipettor be used to simultaneously dispense samples onto the measurement pedestals 3 2 Section 3 General Operation Sample Recovery One of the advantages of the sample retention system is that samples can be recovered from
61. ay or skew when delivering the samples Tip It is suggested that this technique be practiced until consistent delivery to all 8 positions is routine and quick Calibration Check Procedure 1 10 11 Use PR 1 to clean and recondition the pedestals as described above This step must be completed prior to beginning the calibration check procedure Select the Calibration Check Module Tools amp Configuration Tab gt Utilities amp Diagnostics gt Calibration Check Add 55 ul of dH20 to each well of one of the 8 well strips provided Do NOT aliquot the CF 1 at this time Use an 8 channel pipettor to simultaneously pipette 1 5 ul of water to each pedestal position lower the arm and click OK to initialize the instrument Use a lab wipe to remove the water aliquots from both the upper and lower pedestals and change tips Tip It is important to use tips with a rigid structure to ensure the tips do not splay or skew when delivering the samples Use a manual 8 channel pipettor to simultaneously pipette fresh aliquots of 1 5 ul of water to each pedestal position lower the arm and click Blank After the measurement is complete use a lab wipe to remove the water aliquots from all 8 upper and lower pedestals and then change tips Enter the Target Absorbance of the CF 1 in the pop up box Target absorbance 350 nm 1 mm pathlength Tip The lot specific target absorbance is located on the CF 1 ampoule label Thoroughly mi
62. button before exiting the User Preferences window Note User preferences are stored in a log file When upgrading to a newer version of the software this file should be preserved If the user preferences do not appear correctly after upgrading to a new software version the log file should be manually copied to the proper directory 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 ND 8000 Datallog files folder lt is strongly recommended that each time a new user account is added or a password is changed the administrator make a copy of the updated file and store it in the c WD 8000 Datallog files folder If the administrator s account becomes locked the up to date copy can be renamed and used as the password log file 7 5 Dye Chromophore Editor The Dye Chromophore Editor gives the user the ability to add additional dyes or chromophores to the list of predefined fluorescent dyes available for use with the MicroArray and Proteins amp Labels modules Predefined dye methods are indicated by a diamond and cannot be modified Absorbance contribution at 260nm and 280nm from the respective dye can be corrected by entering the appropriate decimal correction in the respective field when adding a new dye to the list Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes Dye Chromophore List Editor Dye Chromophore L
63. ccnncccccconnccnnnnononononcnnnnncnonnnanonons 5 12 Measurement Concentration Range ooonccccnccccccnnncccnnnnnnnnnnnccnnnnnnnnnnnos 5 12 Proteins amp Labels A E E EA 5 14 Fluorescent Dye Selecon reri ana a ei 5 14 Measurement Concentration Range ooooccccncccconnoncccnnncnnnnnannnnnnnnnnnnanos 5 15 Measurement Concentration Range cssseeeeeeeeeeeeeeeeeeeeeeeeeaeeeees 5 17 BCA Assay Sample Preparation cooooonncccnccccononnnccnnnccnononnnnonnncnonnnans 5 18 Prote A a a a T estat 5 21 Measurement Concentration Range ooooccccncccccnnnncccnnncnnnnnancnonnnnnnnnnnos 5 21 Modified Lowry Assay Sample Preparation c ccssssseeeeeeeeeeeeeeees 5 22 io A Ae eel el ee els 5 25 Measurement Concentration Range ooooccccnncccnnnnnnccnnncnnnnnancnnnnnnnnnnnnos 5 25 Bradford Assay Sample PreparatiON oooccccncccnocnnncccnnncnnnnnanccnnnnnnnnnnnos 5 26 Protein Pierce 660 NM oococcconcnnococnnconoconoconcnconononconanononnnnnnonanannnnaranennnnnos 5 29 Measurement Concentration Range ooooccccncccccnnnccccnnncnnnnnancnnnnnnnnnnnnos 5 29 Making Pierce 660 nm Protein Measurement ooccccccccccnncccccnnnccnnnnnns 5 30 GelCulUres tar dia 5 33 Cell Suspension CONCEenNtratiONS ooccccnnccccccnonccnnnnccnnnonnonnnnnonnnnannnnnns 5 34 Sample Homogeneity ooooonncncccnnnnnccccccccnncononnnnnnnnnnnnnnnononnnnnnnonnnnnnznnnnoos 5 34 6 User MethodsS isis ade 6 1 Method Edi ae haces 6 1 Greate New Metode osiin ii
64. ck compound residue is removed and an aliquot of water beads up on the surface Sample Loading Hints Position the instrument at an angle that will allow for optimal use of the pipette guide Ensure the NanoDrop 8000 is not situated near an air vent or an exhaust fan from a nearby instrument Use a small volume 0 5 10 ul manual 8 Channel Pipettor to load both the dH20 and the CF 1 aliquots Tip The use of electronic pipettors is not recommended for this procedure Use shorter more rigid pipette tips such as the 0 1 10 ul low retention tips from Fisher cat 02 717 134 for optimal results The tips MUST fit tightly on all 8 positions of the pipettor Always change pipette tips after each set of measurements Ensure that adequate volumes of sample are being pipetted onto the center of each pedestal Discharge and Touch Sample Delivery Method Practice It is very important to deliver the CF 1 aliquots to all pedestals in a single motion It is highly recommended that the steps below are practiced using water before starting the calibration check procedure 7 8 When the pipette tips are close to the measurement pedestals discharge the fluid and allow the drops to hang on the end of the tips Gently touch the droplets to the pedestals and allow them to be pulled off the tips and onto the pedestals by surface tension Tip It is important to use tips with a rigid structure to ensure the tips do not spl
65. ctors will be displayed on the acquisition screen when operating in either the Single or 8 Sample mode The results of each additional formula will be displayed on the acquisition page when generated in the single sample mode but will not be displayed on the data 8 Sample mode The additional formula results will be available in the data report and archived data for both operation modes Automatic Path Selection If selected the software will automatically switch from using the 1 0 mm pathlength to the 0 2 mm path when the absorbance value of the specified analysis wavelength reaches a threshold of 1 25 6 5 7 Tools amp Configuration Archived Data Sample data from all application modules is automatically stored in archive files and can be opened by either the integrated Data Viewer software program or spreadsheet programs such as MS Excel Archive File Creation Every time an application module is started an application specific archive file is created for the user that is logged in All measurements made by the user in that application module for a given calendar day are stored in a single archive file These files bear the name of the respective application module with the date appended For example an archived file entitled Nucleic Acid 2007 03 21 nd8 corresponds to Nucleic Acid data from the software session that began on March 21 2007 A unique file extension nd8 has been given to these files to enable automatic startup w
66. dard cuveite spectrophotometer Applications UV VIS spectrophotometry is simple for samples as small as 1 ul using the NanoDrop 8000 Spectrophotometer The small sample requirement and ease of use make the NanoDrop 8000 Spectrophotometer ideally suited for measuring e Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng ul dsDNA without dilution e Fluorescent dye labeling density of nucleic acid microarray samples e Purified protein analysis A280 nm up to 100 mg ml BSA Expanded spectrum measurement and quantitation of fluorescent dye labeled proteins conjugates and metalloproteins Bradford Assay analysis of protein BCA Assay analysis of protein Lowry Assay analysis of protein Pierce Protein 660 nm analysis Cell density measurements General UV Vis spectrophotometry Custom methods Operation Up to eight 1 ul samples are pipetted onto the sample pedestal using a low volume multi channel pipettor Each position is actually the end of a fiber optic cable the receiving fibers A second set of fiber optic cables the source fibers are brought into contact with the liquid samples causing the liquid to bridge the gaps between the fiber optic ends The pathlengths are automatically controlled to 1mm and 0 2 mm paths Readings are acquired through sequential measurement across the 8 positions A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to ana
67. data is stored in the archive file at c ND 8000 Data and may be exported to an alternate location To open these reports go to the C ND 8000 Data Reports folder and right click on the file of interest Some key options useful for the Report page are accessible through the Report tool bar drop down ND 8000 Data Viewer vi File Configuration Data EAMA Help Configure Report Plots Repor Sort Report Report Name Save Report Format Load Repor Format Plate Well Sa ID Save Report Load Report Print Report Testtype Report Full Mode Ignore oy g ul A260 A280 260 280 260 230 Constant Cursor Pos Choosing the Configure Report option brings up the following box 7 5 Report configuration editor Select the report columns to display and the order thatthe columns should appear Change the order by selecting and dragging a column header string to the desired position Report columns Plate ID A Vell Sample ID Date Show All Time nq ul Aden A280 260 280 260 230 Constant Cursor Pos Cursor abs Delete hea _Delete_ Measurement Type v Selecting OK will return the user to the Report page displaying only the columns of interest Additional options include e Sort Allows users to sort data by column example by date or sample name and by either ascending or descending order e Save Report Format Saves the current repor
68. decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach solution 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 9 1 Section 9 Maintenance and Warranty Compatibility appendix A final cleaning of all surfaces with de ionized water is also recommended after the user s last measurement Note Do not use a squirt bottle to apply bleach or de ionized water Rapid Reconditioning of the Sample Retention System The Bradford reagent as well as other buffers containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form well with 1ul samples 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 Calibration Pathlength Accuracy Calibration Check It is good practice to verify the pathlength accuracy of the instrument every six months using the CF 8 Calibration Fluid Kit Refer to the calibration check instructions under the Diagnostics amp Utilities heading in section 7 for additional information Wavelength Each time the software is started the wav
69. e concentrations are 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 Eile Standards Help Entrega Dotat 4242007 1017 AM Rotim to Sample Acgurston 1000104 O 18 4 4 j ston or delete repbcoies mami Ave Abs Abs 5 2 Abs 3 0000 0019 B Standard t 012 005 C Standard 2 0250 00 er Standard J 0500 0153 7 Starcard 4 1000 024 i 025 ve Standard 2000 040 0 402 04 0412 E 4 4 4 t ve G Standard 6 4000 0633 063 063 0 061 A Lao jus jos ja tasr ioa To BCA Standard Curve using a 20 1 Standerd 7 8 000 Sadard Cuve Abtorbence Specrs reagent to sample volume 0 2 8 0 mg ml Double Chck on any row to change te con 1 Stenderd Curve Type Polynormel 2nd order Feequored 0 9368 0 38 40 45 50 55 60 6S 70 75 00 mari BCA Protein Sdandard Corro Fie Sinnarda Help _PenPoge oe Dad peed da Ratan to Sample Acque os TOT ZOON 01008 008 013 oae 003 BCA Standard Curve using a 1 1 reagent to sample volume 0 01 0 20 mg ml Senden Curr Abrorbonos Specii Dardani Ciro Typa Exiting the BCA Module It is recommended that you process all of the unknowns to be assayed
70. e Sample Mode ooocccccccccccooccccnccccnnnnoo 3 6 Functions Specific to the 8 Sample Mode oocoooccnccccccccnnccccnccnnconononos 3 7 Standard UI CS sto a E 3 8 4 Sample ID Entry Options cccceeeeessesssssseeeeeseeecenseeeeeeeeeees 4 1 Sample ID entry Single Sample MOde cccccceeeeeeeeeeeeeeeeeeaeeseeeeeees 4 1 Configuration Options for Single Sample Mode ccccccesseeeeeeeeees 4 1 Sample ID entry 8 Sample Mode oooccccccccccccconnccnnncccnnnonncnnnnncnnnonanonons 4 2 Configuration Options for 8 Sample Mode csseeeeeeeeeeeeeeeeeeeeeeees 4 4 Sample Position lllumMiNator oooccnccnnnccccnconncnnnnccnnnonnnnnnnncnnnnnncnnnnnos 4 5 S Standard Methods iia alas 5 1 MAMA EEA E EAA te Meu 9 1 Nc SIC ACIOS a is 5 2 Sample Volume Requirements cccccccccccccccncnccnnnnnnnnnnnnnnnnnnncncnnnnnnnonos 5 2 Measurement Concentration Range occccccccccccnnnnnnonnnnneneneneneneneneneninines 5 2 JGC acVoar E E E mameeces arene 5 4 A E E E 5 5 Sample Volume Requirements ccccccccsssseeecceeeeceeeeeeeeeeeeeseaaeeeeeeeees 5 5 Measurement Concentration Range seececeeeeeeeeeeeeeeeeeeeaeeeeeeeeees 5 5 IGOR IVini eTA E 5 8 Fluorescent Dye Selection Sereen ENa 5 8 Measurement Concentration Range oooccccccccccononcccnnncnnnnnnnccnnnnnnnnnannnonos 5 9 Oligo Calcula e a cuide nel Rua e ice 5 10 SA A 5 12 Sample Volume Requirements oooocc
71. e formed so that the gap between the upper and lower measurement pedestals is bridged with sample Note It is not necessary to have liquid on all 8 positions to make a measurement Field experience indicates that the following volumes are sufficient to ensure reproducibility e Aqueous solutions of nucleic acids 1 ul e Purified protein 2 ul e Bradford BCA Lowry or Pierce Protein 660 nm Protein Assay 2 ul e Microbial cell suspensions 1 2 ul It is best to use a calibrated precision pipettor 0 2 ul with low retention precision tips to ensure 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 surface tension characteristics or pipettor accuracy a 2 ul sample is recommended Use an 8 channel pipettor with good fitting tips when loading multiple samples to minimize evaporation due to delays in sample loading If the tips splay or skew when touching the pedestal surfaces please use an alternative brand or style with a more rigid tip structure It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Sample Carryover Prevention of sample being retained on the NanoDrop 8000 Spectrophotometer s measurement pedestals is easily addressed Simple wiping of the upper and lower measurement pedestal with a dry labor
72. e standard points depending on the polynomial degree selected 9 19 Section 5 Standard Methods BCA Protein Standard Curves File Standards Help User Default 4 3 2007 2 43 PM Return to Sample Acquisition Measurements Table Double Click on any row to change the concentration or delete replicates Y maja ave abs Abs 1 Abs 2 Absa Absa Abs 6 measure Active m A Reterence 000 0008 0007 0007 0002 omo 0015 Active B 6 Stonderd 020 005 ooa oosa oos 00 009 Active B C Stenderd2 040 0093 o103 0099 oosa o100 0088 Active MI D Stondara3 050 0120 0117 o120 0123 0123 0118 Active B E Stenderaa 100 0210 o211 0205 0215 0213 0207 ace E _F stondaras f 20o 0 355 0357 0354 0355 0356 0352 Active B G Stonderas 400 0591 0593 osa 0593 0590 osae Active B H Standera 600 0923 092 0 322 Standard Curve Absorbance Spectra HA AA Che AA hce YY oo YY Ch E hF A Ch G MH IS 450 480 500 520 540 560 580 600 620 640 660 680 700 720 750 Wavelength e Measure Samples Once a minimum standard curve has been established the standard curve indicator will change from gray invalid to green valid allowing the user to start measuring samples The designation of Valid only indicates that the minimum requirement for a 2 point curve has been met Sampl
73. econnect the instrument If this occurs the USB cable will need to be disconnected reconnected before selecting Retry You can confirm that the power management settings are correct by opening the Power Options Properties page by choosing Start gt Control Panel gt Power Options The System Standby and System Hibernate should be set to never for the Plugged In column Power Options Properties ax Power Schemes Alarms Power Meter Advanced Hibemate E Select the power scheme with the most appropriate settings for this computer Note that changing the settings below will modify the selected scheme m Power schemes Save As Delete m Settings for Home Office Desk power scheme When computer is Plugged in Running on batteries Turn off monitor After 20 mins y after 15 mins y Turn off hard disks Never y After 10 mins y System standby Never b After 30 mins System hibernates Never After 45 mins e Defective USB Port on PC If your instrument operates properly most of the time but the Connection Error appears intermittently it could be caused by the USB port on the PC If this occurs install the software and operate on another PC Ifthe error does not occur on the second PC it may be necessary to replace the USB card on the original PC 8 3 Signal Error Signal Error This is most likely caused by Sample surface is
74. econtamination of Measurement Pedestals ccoooncococonnnconono 9 1 Rapid Reconditioning of the Sample Retention System 9 2 Cala e ae ld le LS 9 2 Pathlength Accuracy Calibration Check cccccccooccncccccocconccnonncnnconanonos 9 2 TAY Fe AVC A IO ene Or 9 2 A A O eee CoE San E 9 2 10 AD DONGICCS ici 10 1 Instrument Specifications a ss 10 1 Blanking and Absorbance Calculations oooooooccccnnnccccccnnnnccncnnnnnnnnnnnnnnos 10 1 Concentration Calculation Beers Law ccoocnnncccnncccccconnonononocononanonons 10 1 A ne ara re Oe Meee eee ee tere ane nee ree 10 1 Nucleic ACOS re is 10 2 Section 1 Overview 1 Overview Instrument Description The Thermo Scientific NanoDrop 8000 Spectrophotometer is a full spectrum 220 750nm instrument that accurately measures up to 8 individual 1 ul Samples in one measurement cycle The software allows the user to measure samples using either the full 8 position mode or a convenient single sample mode The NanoDrop 8000 utilizes the same patented sample retention technology employed on all NanoDrop instruments The surface retention system holds the sample in place eliminating the need for cumbersome cuvettes and other sample containment devices Clean up is accomplished in seconds In addition the NanoDrop 8000 has the capability to measure highly concentrated samples without dilution 50X higher concentration than the samples measured by a stan
75. ected Plot drop down box which will also display the legend The selected sample will show up as a bold plot line e 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 e 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 e 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 e Movable x and y axis Available for all data types If the cursor is out of view in either direction rescale the axis by typing over one of the outer limit numbers The cursor absorbance information displayed at the bottom of the page is determined by the position of the movable cursors The movable X determines the baseline from which the peak of the Y position is calculated Reset Baseline will reposition the x axis back to zero Report Page The Report page displays the data for selected samples in a table format The user may modify column configuratio
76. elected via the units drop down box The units will default to mg ml each time the software is opened e Show Report formatted for 200 samples although the buffer size can be modified Baseline Type This application module has two user selectable Baseline Type options The default setting is set to normalize the display spectrum at 750 nm 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 280 nm absorbance value is normalized at 340 nm unless the user elects to turn off the normalization 9 16 Section 5 Standard Methods 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 that a standard curve be generated before unknown protein concentrations can be determined 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 CuSO4 utilized in the assay are available in kit form from numerous manufacturers Follow the respective manufacturer s recommendations for all standards and samples unknowns Sample Volume Requirements The presence of surfactants o
77. elengths are auto calibrated based on known peaks in the xenon lamp spectra No calibration is required by the user 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 Additional one two and three year service plans are available Additional information may be found on our website 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 very erratic or stop altogether Contact Technical Support or your local distributor is you suspect your lamp may need replacing 9 2 Section 10 Appendices 10 Appendices Instrument Specifications e Sample Size 1microliter Sample Number up to 8 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 3 at 0 74 absorbance at 350 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 20 seconds Dimensions footprint 24 x 32 cm Wei
78. en running a custom method User Preferences File Help User Default Microarray Proteins amp Labels Protein 4260 General Settings Reports Data Exporting Nucleic Acids UV Vis Auto Reporting Nucleic Acids Protein 4280 Select Custom UV Vis Protein BCA Report Format Microarray Protein Bradford Proteins amp Labels Protein Lowry Single Sample O Cell Cultures Y Protein Pierce 660 nm 8 Sample Default Reports Module Custom Report Nucleic Acid Standard Nucleic Acid report format nt MicroArray Standard MicroArray report format nf UV Vis Standard UV Vis report format nte Protein A 280 Standard Protein A 280 report format nf8 Cell Culture Standard Cell Culture report format nf Proteins amp Labels Standard Proteins amp Labels report format nf BCA Protein Standard BCA Protein report format nf Bradford Standard Bradford report ftormat nf Lowry Standard Lowry reportformatnfa Pierce 660nm Standard Pierce 660nm report format nf8 Custom formulas nf K Auto Reporting Users may choose to select the Auto Reporting option for any of the application modules The auto reporting option allows data to automatically be saved to the report for all samples Users may choose this option under the Reports tab by selecting the corresponding box next to the modules listed under Auto Reporting Save the auto reporting functions by clicking on the Save amp Exit
79. escent 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy 5 8 Section 5 Standard Methods Measurement Concentration Range The NanoDrop 8000 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 Detection Typical Reproducibility Limit minimum 48 replicates pmol ul SD pmol ul CV Cy3 Cy3 5 Alexa sample range 0 25 4 pmol ul Fluor 555 and Alexa 0 25 Fluor 660 l sample range gt 4 0 pmol ul 2 5 sample range 0 17 2 4 pmol ul 0 17 sample range gt 2 4 pmol ul 2 5 Alexa Fluor 488 and sample range 0 50 8 0 pmol ul Alexa Fluor 594 0 50 l sample range gt 8 0 pmol ul 2 5 sample range 0 42 6 0 pmol ul 0 42 sample range gt 6 0 pmol ul 2 5 Alexa Fluor 546 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be set
80. est marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 2 Section 5 Standard Methods Unique Screen Features T Huchet Acid E 2 File Edit Configuration coro Help Menzure Blank Fieblank Recording ShowPiepor User Dietoutt Dete Time 9 2008 221PM Et Piste ID 425 ng u Measurament completa ee Sample Type DAA All Active On Off nm 260 Limite mel Active m 5 1 A A Sapt 1 miaa AA ADA AI naiul Sample ID 440 rgd A 200 4749 3600 1 05 a 215 puun a a J Actve m 5 1 Fa A At Sample 1 rm Tobe AASE ADR ASE aa k Semple lD HO ngid Amo 4705 60 260 16 v2 213 n Aces m 1 ie Sy Ci Sample 1 rata 4754 AQ 2754 nahi Sample ID HO ngiu w Amol 4725 200l 105 oe 217 TN za J Aawe m E i si D3 Ssmplet 1 mias AN AQ AN ha p hn comple HO rut A en ATR 260 7200 15 Soe 215 Aca E 1 Lf i EJ SampleH 1 mise B782 Agel 4 i Gs 3 12 oat tia h AJ 47r aita 107 W02 215 mi 24000000000000 Activo m E 1 AA F3 Semele md abs 8763 Azo 8763 50180000 O OODODODO impen Honga A A a aa E ue 213 E 000000000000 Sampin AD rl A280 4 70 PRIOR 187 SO 213 Campo 100 440 rod naful naful 0400000000000 Active mj E 1 Mon GI Semple 1 rmiabt 00004 TA oz a AN HI sampat
81. ethod parameters Step 5 of 5 Name Method name l Alexa 488 Protected optional Note A method that is protected can only be modified when accessed under the user account from which it was created It is recommended that a method be created from a password protected user account rather than the Default user account to ensure that methods are not inadvertently modified 6 3 Section 6 User Methods Edit Selected Method Predefined methods with a black diamond next to them are protected and cannot be edited User defined methods with a black diamond by only be edited the user hat created All other methods may be edited at any time by highlighting the method name and hitting the Edit Selected button All four wizard pages are accessible as tabbed pages when editing a method Edit method parameters MeasurementType Wavelengths Baseline Name The concentration is calculated by multiplying the absorbance A atthe Analysis wavelength by the Factor and dividing by the path length Both the Analysis nm and the Factor are required Quantification Mode c FactorxA b v Factor Value ng ul 1 00E 0 Analysis Wavelength nm 300 Units ngul v Mol Weight g mol 0 00E 0 The user may move to the appropriate window by using the top tabs and edit only the parameters of interest The user may also edit a method from within the acquisition module by using the Edit drop down box Note When edit
82. files 3 plates Data by rows tet Sample Well Position Assignments 1 2 3 4 5 6 7 8 10 111 112 DNA AT DNAA2 DNAA3 DNAAS DNAAS DNAAS DNAA7 DNAAS DNAAS ONAMO ONAM DNAA1Z PONABT DNAB2 DNAB3 DNAB4 DNABS DNABG DNAB7 DNABG DNABS DNABIO DNAEM DNABI2 DNA GT DNAG2 DNAG3 onana DNAG5 DNAG6 DNAG7 DNAG8 DNAG9 DNAGIO ONAGM DNAGI2 DNAHS DNAHTO ONAM DNAHT2 TTL You may choose to skip sample columns in this Plate Set by choosing a Start Column other than 1 default Start Column i a l Define Sample ID File Format The Plate Set selector will allow the user to review that each set of samples fills the screen map in the expected order If there is a discrepancy click on the Define Sample ID File Format button on the bottom right to return to the previous step Manual Entry This option opens the Enter Sample IDs window enabling the user to manually enter samples IDs and number of replicates for each sample to be tested as prompted by the following screen Enter sample Us Enler the Sample Ds wa lhe keyboard or barcode scanner The well position Increments aulomalically wilh each entry Chick on awell to go to thot well Fsamples 96 J 12 DODODODD DODDDOODODOO DODODODODODDO ODPODDDODODO DOPODODODODO DODODODODDDO DODODODODDDO DODODOPODDDO To de select 6 wall setthe replicates 10 0 Position Al E Aopliusalies arma II A D E D E F G
83. 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 USB Flash Drive Port Any standard PC USB flash drive may be used for exporting data Note When using the User Preferences Module to set up a default automatic Export Report destination keep in mind that the flash drive may not always be assigned the same removable device designation 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 NanoDrop 8000 Spectrophotometer is supplied with a 12V power supply Use only the power supply provided with the kit The unit also comes with a grounded power cord Plug this cord ONLY into a properly grounded outlet Use of the instrument in a manner not specified by the manufacturer may impair the protection provided by the supplied power cord and power supply The power supply can remain plugged into the NanoDrop 8000 Spectrophotometer while the instrument is not in use When the instrument is plugged in but not in use the power con
84. g option establishes a new reference blank that is used for the absorbance calculations of subsequent samples The Re blank is only applied to the specific samples selected and re calculates the concentration for those samples respectively Although a new spectrum will be displayed on graph and the previous samples data will be recalculated and saved in the archive file the recalculated data will not be displayed in the current report See the Blanking and Absorbance Calculations appendix for more information on absorbance calculations Start Report Recording All data is automatically archived The user can log measurement results in a active report table as the data is accumulating by using the Start Report Recording feature The default setting has the Recording feature activated for all modules If Start Report is displayed the accumulating data will still be archived but will not be shown in the active report 3 4 Section 3 General Operation Show Report Selecting this button will bring up the Report page which is part of the integrated Data Viewer software A full description of the features and options for the Report page can be found in the section on the Archived Data and Data Viewer User This field displays the name of the user account in use User Guidance Display Box This field displays either instructions or measurement status information as appropriate Units A drop down box on the right side of the acqui
85. 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 9 gt Leninger A L Biochemistry oe ed Worth Publishers New York 1975 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 Located on the bottom of the unit e JPG image of Utilities and Diagnostics module To get this open this module and select OK to initialize the module Select Intensity Check Once the spectrum has been created choose File gt Save Window as shown below Save to your hard drive and email as an attachment to your local distributor or to Technical Support z D 3000 3550 4040 4500 500 540 200 BO 7000 7500 2040 2500 dara Cosfquimsn Dal 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
86. generating a standard curve from a previously saved standard curve file macia E Fl Standard 7 The Standards menu drop down may also be used to load a previously saved curve generate a new standard curve or view the current standard curve See the respective protein assay section for additional details Additional features User Manual The User Manual is accessible from the Main Menu and from the Help menu in all of the application modules It can also be accessed by selecting Start gt Programs gt NanoDrop gt ND 8000 version Print Window A Print dialogue can be initiated from the File pull down menu or by typing Ctrl P 3 8 Section 3 General Operation 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 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 3 9 Section 4 Sample ID Entry Options 4 Sample ID Entry Options Sample ID entry Single Sample Mode Selecting the Sample Loading Mode After the instrument has completed the initialization process the following window will automatically display the options for plate setup Select Sample Load Load Sample ID Manual Sample ID File Entry Cancel Load Sample ID File The NanoDrop 8000 software enables the user to load a list of predefined sample IDs or n
87. ght 3 5 kg Sample Pedestals Material of Construction 303 stainless steel and quartz fiber Operating Voltage 12 Vdc Operating Power Consumption 30 W Standby Power Consumption 3 W UL CSA and CE approval all units e Included in system software compatible with Windows 2000 XP Vista 32 bit and Windows 7 32 bit and 64 bit Blanking and Absorbance Calculations When the NanoDrop 8000 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 The software uses the gene
88. guration Note Access the User Preferences module on the main menu to modify and save preferred default configurations Reports are saved in an nr8 format The user may select specific default report configurations for each pre defined method Only one default report formula is available for all user defined methods Use the drop down Load Report Format to utilize a different saved configuration when running a custom method 7 6 The other two options are meant for reports that are expected to be opened in Excel type spreadsheets To open these reports go to the C ND 8000 Data Reports folder and right click on the file of interest Additional features of the Report page e Method Automatically populated with data method type e Date and Time Automatically populated when report is generated e Report Name User defined designation for the current report e Report Full mode Drop down box defining options for managing reports The user may elect to Print Save or Print and Save a report at any time by using the Report Full Mode drop down box shown below The default setting of 1000 samples per report may be modified by highlighting the box and typing in the desired number Choosing Ignore from the drop down will allow the user to include an unlimited number of samples in a report e Max Report Size Default number is set at 1000 Standards Page The Standards page will display the actual reference standards applied to each particular sample
89. he 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 the Protein A280 section for a detailed description of each sample type e nm 1and nm 1 abs current value of the user selectable wavelength cursor and corresponding 10mm equivalent normalized absorbance at the respective wavelength absorbance The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e Dye 1 user selected dye e Dye 2 user selected dye e Dye 1 or 2 abs normalized 10 mm equivalent absorbance of selected Dye e Dyes can be selected using the scroll arrows or by highlighting the Dye box The respective absorbance wavelength extinction coefficient and 260 nm and 280 nm corrections will be automatically utilized for measurement and concentration calculation The default setting for Dye 1 is Cy3 and Dye 2 is Cy5 See User Preferences under the Tools amp Configuration main page tab to designate alternative dyes as the defaults e Concentration mg ml sample concentration based on the absorbance at 280 nm and the selected analysis constant The calculated concentration is displayed in the units selected via the units drop down box The calculated concentration is displayed in the units s
90. her column A barcode reader can be used to scan in individual bar coded sample names into the txt file The column format enables the user to predefine and load an unlimited number of sample IDs using just one plate file Note Alternatively sample names may be stored in an 8 x 12 array within a txt file The 8x12 format can only load one plate worth of sample ID s at a time 96 samples As there is already a one to one direct correspondence with the on screen plate map when using this format one does not need to use the Define Sample ID File Format button Use the Load Sample ID File button to directly load 8 x 12 array sample ID information Define Sample ID File Format With the exception of the 8 x 12 array format described above before a txt list file can be loaded the Define Sample ID File Format feature should be used to correctly specify which orientation the sample IDs should correspond to well positions on the plate map on the software acquisition page The user is first prompted to select a pre existing plate file from the Plate file folder using the browser dialog box Use the Data Series Type drop down box to select the proper sequence columns or rows Sample names will either fill in and correspond to well positions in Rows first 12 sample IDs will correspond to the A1 to A12 positions the next 12 IDs to the B1 to B12 positions etc or in Columns first 8 sample names will correspond to the A1 to H1 positions the next 8 names t
91. hrough them Further information regarding our compliance with these directives the recyclers in your country and information on our products which may assist the detection of substances subject to the RoHS Directive are available at www thermo com WEEEROHS Patents The sample retention technology used in the NanoDrop 8000 is covered under US patents 6 628 382 and 6 809 826 Other patents are pending 2 Section 2 Initial Set up 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 Microsoft Windows 2000 XP or Vista 32 bit and Windows 7 32 and 64 bit Windows Vista has also been tested successfully with the software The operating software is not compatible with Windows NT 95 98 or ME 800 MHz or higher processor CD ROM drive 128 MB or more of RAM 100 MB of free hard disk space Open USB port the instrument can only be connected via the USB port Microsoft Excel or other spreadsheet program to manipulate archived data optional Unless otherwise specified all screen shots have been generated using the Windows XP operating software Software Installation IMPORTANT PLEASE READ BEFORE INSTALLING SOFTWARE Note 1 The system software must be loaded onto the PC before the USB cable is connected Note 2 Uninstall all previous versions of NanoDrop 8000 software using Add Remove Programs B
92. ic strength of an undiluted sample measured on the NanoDrop 8000 is at the same 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 e Wavelength accuracy of the spectrophotometers Although the absorbance of a nucleic acid at 260nm is generally on a plateau the absorbance curve at 280nm is quite steeply sloped A slight shift in wavelength accuracy will have a large effect on 260 280 ratios For example a 1 nm shift in wavelength accuracy will result in a 0 2 change in the 260 280 ratio Since many spectrophotometers claim a 1 nm accuracy specification it is possible to see as much as a 0 4 difference in the 260 280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are both within wavelength accuracy specification e Nucleotide mix in your sample The five nucleotides that comprise DNA and RNA exhibit widely varying 260 280 ratios The following represent the 260 280 ratios estimated for each nucleotide if measured independently Guanine 1 15 Adenine 4 50 Cytosine 1 51 Uracil 4 00 Thymine 1 47 The resultant 260 280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260 280 ratios for the four nucleotides present It is important to note that the
93. ie G 250 and the Coomassie Plus Protein Assay kit from Pierce preliminary results show the Coomassie Plus Protein Assay produces better reproducibility amongst triplicate readings than the regular G 250 stain when reading the samples directly after the optimal incubation time 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 8000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay Both single and multi point standard curve generation is incorporated into the software A standard curve can be developed using a reference Bradford reagent only no protein and a single replicate of one standard There is 9 26 Section 5 Standard Methods no set order in which standards must be run The multi point standard curve generator displays a maximum of 5 replicates for each of 7 different standards The following box will appear after the module initialization is complete Choose Standards Source Standards are required Choose the Standard Curve source Load Standard New Standards Curve Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below On DEA SS 1 000 concentrations and curve type for Active
94. ient of 6 7 at 280 nm for a 1 10 mg ml BSA solution IgG reference Unknown sample protein concentrations are calculated IgG v using the mass extinction coefficient of 13 7 at 280 nm for a 1 10 mg ml IgG solution Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution Sample Type Other protein E amp MW 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 999 X 1000 and maximum value for e 1000 1 00 M W is 9999 X 1000 Mol Weight kDa 1 000 Sample Type Otherprotein E1 v User entered mass extinction coefficient L gm cm for a 10 mg ml 1 solution of the respective reference protein Ext Coeff E 1 L gm cm 10 00 e nm 1 and nm 1 abs current value of the user selectable wavelength cursor and corresponding 10mm equivalent normalized absorbance at the respective wavelength absorbance The 9 6 Section 5 Standard Methods wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e A280 10 mm Path 10 mm equivalent absorbance at 280 nm for the protein sample measured e A260 280 ratio of sample absorbance at 260 nm and 280 n
95. ies in the Account Management module establishes whether individual user passwords will expire and if so after how many days Maximum Password Attempts 100 Minimum Password Length 0 Password Expiration enabled P A Default Expiration Days 360 Passwords log file This file contains the User ID amp password for all accounts and is readable only by the software It can be found in the c ND 8000 Datallog files folder It is strongly recommended that each time a new user account is added or a password is changed the administrator make a copy of the updated file and store it in the c ND 8000 Datallog files folder If the administrator s account becomes locked the up to date copy can be renamed and used as the password log file User Preferences Each user has the option to configure a number of settings in the various application modules The user preferences options for each application module are self explanatory and include options applicable for that module Some key features include The General Settings tab allows the user to either select or deselect as default settings the options of 7 3 e Requiring sample ID s before a measurement is taken e Auto advancement to the next set of samples i e move to the next column on the 96 well visual e Requiring the user to confirm that they want the Data Viewer to close when closing out of an application User Preferences File Help Default Microarray Protei
96. include e File Allows the user to define the page set up for printing out the spectra the report and the standard curve This drop down also allows the user to save the window as a jpg e Configuration Options controlled by this tool bar function include Auto Scale Include graph in printout and Include standards in printout e 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 e Reports This tool bar function allows the user to select columns of interest to be included ina report See following section on Reports Page for details on additional drop down box options e Print Window The current Plot Report or Standards screen may be printed by selecting Print Window or CTL P e Save Window Saves files as JPGs Import Page To select samples for viewing select Import Samples from the Data menu bar drop down options from 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 below 1 2 Sari la sidlinciad lo Aa at current ae branch of e Beinn tpn Features include e Import folder Used to select folder from which data will be imported Folder selection must be at the level of user or higher may
97. ing is complete the changes must be saved in order to be implemented View Selected This button allows the user to review the parameters of a method but will not save any changes 6 4 Section 6 User Methods User Method Acquisition Screen Example custom File Eda Configuration Help Mies Alark i Hr hl nk Recording Shoa Popat Lger tanl Dati Teng 3722008 945 AA Eri PD Make new BLANK Measurement Auwomane Poth Selection E All Acie Cont nl 260 E Limits ngid Active ef i AT F U Tanie N 1 pm late 1118 SSR 1 ng ul Sample ID Sample ADO 1 110 Agil 01602 555 th Anciysic vvewalangin SBH Acie 7 a A e Set 1 ren Tate 1 136 DE 1 ng ul Factor 50 00 Sample ii Sampir i AXD 1 138 Aso 00633 560 2 Mol Weight g mol 0 000 bates n oR EH Saet a miae 1 158 DA LA act Sample Sama Aal 1 158 Agen MRSA 573 1 K 2 a el 1 Semen 1 miae 1 171 PSN Lia nail Sample If Sample 4 a ce gt 1 131 Aven 05613 565 6 Activ ET 5 eC Sangin 1 relate 1 069 PvE 1 76 ogui 2 Sample 1D SemeleS ace 1 065 aceon nam STE Holin Ti He Pi O Sarge HH 1 emote 1155 TLR 143 ng ul Same ID Sangki LA A 1 155 Aaa 00631 Elia Pt Gal 5 fm F F H Semple ID Sampin Awn 1119 A 0 614 ooo Mire 0O a 1 ih HT Sample tt 1 pm ae 103 0 1 ng ul Sample ID Sampin i Ace Lora AD 600 536 7 The analysis wavelength and associated fa
98. ion Cleaning instructions the calibration check procedure as well as a table of calibration check tolerances may be accessed via the menu bar Help drop down feature Intensity Check The Intensity check is used for troubleshooting purposes The below image is representative of a typical spectra Refer to the chapter on Troubleshooting for additional information 7 1 Se ol Number UVSOEGITSCRE Fedesini 1 Detecior fins 34 LU Si pach 421000 _ e Intensity Chock Ong Pedistol lA mig 8 mg ly 6 Cloor apii ami gt z integration Timer pr ar i 1 i i i 1 l fi Ml i i 200 2000 2010 500 2500 2760 206 200 2000 3100 200 1160 mii Wii Wevelasq nmi Wslbl Spectra Wieolongih Avccunescy hax Erie Ofni 04 anon Panke Manternd ad nm 4945 260 7 nm 2605 E 625 mm 3671 TeS2m00 3 5 ilzam ee y l i i 4 i i p 1 i i i i 200 200 3500 4060 4500 200 500 500 6500 7000 7500 000 500 A nn Contgurotion ADETEN Account Management The Account Management application provides options for directing where specific data files are archived allowing users to segregate their data into personal folders The Account Management module is accessible to the administrator only User Access Manager Actions All Users Full Name Default Default user 0 Active NotLocked NotExpired Never Administrator Administrator NotLocked NotExpired Never
99. ist ele A oe Name 1 M cm nm g Mol 260 nm 280 nm factor factor 07 150E 550 oo0E ooo 000 Below 05 2508 650 o008 0 000 000 Alexa Fluor 488 IOE 4 495 0 00E 0 0 00 0 00 Alexa Fluor 546 o4e 5 556 000E 0 ooo ooo eee Alexa Fluor 555 15085 555 oooE o ooo ooo selected Alexa Fluor 594 73074 690 0000 ooo 000 Alexa Fluor 647 23315 650 oo0e 0 ooo 000 Alexa Fluor 60 T325 oss oo0es0 ooo ooo O35 rs0E s 681 o00e 0 ooo ooo 055 es0e s 67 000E ooo ooo Test roooe o 220 0000 ooo 000 i tf Note predefined dyes are indicated with a diamond and cannot be modified 7 6 Section 8 Troubleshooting 8 Troubleshooting Error Codes Unrecognized Devices Instrument Peripheral Control Device or LED Position Indicator Not Found These errors might appear upon software startup and usually indicates that either the power supply or the USB cable is not properly connected or the specific drivers are not loaded properly To troubleshoot do the following 1 Check that the power supply is connected to the instrument Confirm that the instrument is getting power by observing that light can be seen through the USB opening on the rear of the instrument 2 Confirm that the USB cable is connected to both the PC and the instrument Note There are internal USB drivers in addition to the USB cable When attaching the USB cable please wait at least 30 seconds for the
100. ith the Data Viewer see the description of Data Viewer later in this section 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 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 is stored based ona 1 0 cm 10 0 mm path For MicroArray UV Vis BCA Protein Bradford Lowry and Cell Culture application modules the data is normalized to a 1 0 mm 0 1 cm path For data from all modules a column entitled Measurement Type is included For each measurement this column will contain Measure Blank or Reblank If the value is Measure then the values in that row are from a normal measurement that has utilized the stored blank value If the value is Blank it indicates that the measurement Is the initial blank recorded If the value is Reblank it is the re analysis of the previous measurement with a new blank Ed Microsoft Excel Nucleic Acid 2005 11 01 v3 2 ndj Read Only Ed ie Edt ew freet Format Took fats Window ACTI Heb Adobe FOr r SA PEN A a E y 0 Arial 210 BIO EEN S or MO F fe Module em e mew E F 6 H 1 J 3 TK IT L MJ H J Moduda Nucleic Acid Ua _2 Path Dl mri 3 Solwa 3 2 0 A Firan USEAN 2 41 3 HOS Al s
101. licates specified in plate set up Units c Protein A 280 File Edit Configuration Help Measure Blank Recording ShowRepor User efault Date Time 8 15 2008 8 55 AM Exit Plate ID Measurement complete Sample Type ABST mg ml v All Active OwOft 11280 Units mg m Actve 1 Ra Sample 2 ren labs 10 16 260 280 1512 oan Sample 1D tes A280 10 16 eae and Sample The indicator to the left of the spectra indicates the number of replicates originally called for when sample names were entered in manually or by loading a plate file The Sample located to the right of the spectrum is activated when a sample measurement is being recorded It indicates the replicate number of the last sample processed for a particular well and increments with each successive measurement Expanded Sample Spectrum View The user may display an expanded view of a single spectrum by clicking on the spectrum of interest This view will display one movable cursor with the respective nm and absorbance reported in the boxes at the bottom of the screen Note Data is archived as the measurement is made Changing the cursor position after the data is archived will not change the data files Moving the cursor via the expanded view will not change the selected wavelength positions on the main acquisition page 3 Section 3 General Operation Al mar Standard Curves A standard curve is required every time a BCA Lowry Bradford or Pie
102. ll measurement surfaces with de ionized water is also recommended after the user s last measurement Note 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 solution 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 3 1 Section 3 General Operation It is recommended that a final cleaning using de ionized water be made if using a sanitizing solution on the measurement surfaces to ensure any residual solution is removed Note Do not use a squirt bottle to apply bleach or de ionized water Rapid Reconditioning of the Sample Retention System The Bradford reagent as well as other buffers containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form well with 1ul samples 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 Sample Size Requirements Although sample size is not critical it is essential that the liquid column b
103. lyze the light that passes through the samples The instrument is controlled by PC based software and the data is logged in an archive file on the PC The NanoDrop 8000 is designed only for indoor use under the following conditions e Temperature 40 100 F 4 4 37 8 C e Humidity 10 90 Safety The NanoDrop 8000 is supplied with a 12V power supply Use only the power supply provided with the instrument The unit also comes with a grounded power cord Plug this cord into a properly grounded outlet Use of the instrument in a manner not specified by the manufacturer may impair the protection provided by the supplied power cord and power supply The power supply can remain plugged into the NanoDrop 8000 while the instrument is not in use When the instrument is plugged in but not in use the power consumption is 5 W and the flash lamp is 1 1 Section 1 Overview not energized The instrument does not utilize a power switch It is recommended that the instrument not be positioned in a way that makes it difficult to unplug the power supply from the unit or the wall WEEE Compliance This product is required to comply with the European Union s Waste Electrical amp Electronic Equipment WEEE Directive 2002 96 EC If compliance is required the instrument is marked with the following symbol a We have contracted with one or more recycling disposal companies in each EU Member State and this product should be disposed of or recycled t
104. m e Concentration mg ml sample concentration based on the absorbance at 280 nm and the selected analysis constant The calculated concentration is displayed in the units selected via the units drop down box The calculated concentration is displayed in the units selected via the units drop down box The units will default to mg ml each time the software is opened See the Concentration Calculation Beer s Law in the appendix for more details on this calculation e Show Report formatted for 200 samples although the buffer size can be modified 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 unless the user elects to turn off the normalization 9 7 Section 5 Standard Methods 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 8000 Spectrophotometer measures the absorbance of up to 2 fluorescent dyes allowing detection at dye concentrations as low as 0 2 picomoles per microliter Fluorescent Dye Selection There are currently a number of 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 8000 software using
105. measured is homogeneous Field experience has shown that samples containing large molecules such as genomic or lambda DNA are particularly susceptible to this phenomenon Note Larger volumes used by cuvette based spectrophotometers will minimize or mask the effect of sample non homogeneity e 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 same buffer or solvent the unknown samples are in onto each of the lower measurement pedestals and then lower the sampling arm into the down position 3 Click on the Blank button When the measurement is complete wipe off the buffer from all pedestals 4 Select All Active On and analyze a fresh aliquot of the blanking solution on all pedestals using the Measure button F1 The result should be 8 spectra with relatively flat baselines near zero 8 8 5 Wipe the blank from all upper and lower measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum varies no more than 0 005 A 1 mm path from the baseline Note The Nucleic Acid A280 and Proteins amp Labels modules display the absorbance values normalized to a 10 mm path so the effective variance should be 0 05 abs from the baseline 6 Reload the plate
106. multiple USB devices to be installed and recognized before opening the software 3 If the cable is connected properly but the Instrument recognition error persists open the Windows Device Manager by either right clicking on the My Computer icon on the desk top or selecting Start gt My Computer right click gt Manage gt Device Manager gt NanoDrop Devices aj Computer Management Local Batteries i System Tools Ha Biometric 80 Event viewer 88 Bluetooth Shared Folders P Computer Local Users and Groups See Disk drives E Performance Logs and Alert E Display adapters g a Device Manager 22 DYDICD ROM drives y Storage IDE ATAJATAPI controllers e fe Removable Storage IEEE 1394 Bus host controllers Disk Defragmenter Keyboards Disk Management Mice and other pointing devices Ta Services and Applications b Modems El Monitors HanoDrop Devices NanoDrop LED Position Indicator lt _o ND 8000 Peripheral Control Device ND 8000 Spectrophotometer 4 88 Network adapters E PCMCIA adapters q Ports COM amp LPT 4 If an unknown device a yellow exclamation point or a question mark appears next to one of three expected NanoDrop devices manually uninstall the device by right clicking and selecting Uninstall from the options displayed 5 Disconnect the USB cable first and then disconnect the power supply from the NanoDrop 8000 Reconnect the power supply wait 5 seconds and the
107. n Suspension The Cell Cultures module displays the sample spectrum from 250 nm to 700 nm The software will display the absorbance data for the frequently used wavelength for monitoring cell suspensions 600nm in addition to displaying the value for a second wavelength of interest By selecting Measurement Limits from the configuration drop down menu minimum and maximum absorbance limits can be set for the user selectable wavelength cursor position These limits cannot be set as a default and must be defined each time the application module is opened Sample absorbances that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The absorbance values will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 33 Section 5 Standard Methods Unique Screen Features TC Cudira 2 File Edit Configuration Srcod Help Mesure Blank i Reblenk Recording Show Repor User Deteult DeTime 2003 327 PM Em Plate ID Inr Spec Measurement completa a All Active Ona amt 340 Actin 8 1 Al_ Sane tt 4 mmia 1477 Aen nna Beira E 1 N Al Sampat 1 mmia 1453 ARD nam Acton 8 1 Cl Sample 1 mata 11450 A Senge ID e
108. n Tale BTA AGH 6764 TN Sample lO 440 mg M a AO 42 gt FRED 1 eM 217 cps Active m 4 1 Poli DA Sample 1 rodada 87E ADR ATA Mn ngful Semple it anng aan ar awol Lee 2000200 219 Era bae m y 1 AN EJ Semple ay ren Tabs 8782 Age BE Ber Sample D 440 rgd Ami AE NAO 1 87 pones EA E 1 3 E 5 0009000000000 t E A E _ 8768 AA 59409 0000000O0D O seein ngid am ame 260 200 1 66 zum 219 Bea Active m 1 GJ Swapit i 0440000000000 ve m Ma mapie i nm labs Baw 2260 6 004 sere E SSOCOCCOCOCOOD Free lb imu azo 4710 ama 18 ZA 2 19 e 0O00000000 Acton m oH 1 ats H3 Sepen 1 mmia enc AR RRI i cee 0 000000000 Semple ID daing ia a Azan 4715 i 219 fou HsPSSOOCOCO0CDOD a eee IA a 20 0 emsa iare Sample Plate Map The on screen sample plate will be automatically populated if a Sample ID file is imported Alternately the user may manually type in a Sample ID or other identifying descriptor The Sample Position Illuminator allows the user to visualize the row of a standard 96 well micro titer plate that is to be sampled for measurement This lighted guide corresponds to the sample status color code displayed on the software screen and the pattern of illumination is determined by plate configuration at set up Status The Status button will turn green during a measurement cycle Indicates actual number of replicate measurements made Indicates number of rep
109. n arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e A260 absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm path Note This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length e A280 sample absorbance at 280 nm is 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 e 260 280 ratio of sample absorbance at 260 nm and 280 nm The ratio of absorbance at 260 nm 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 9 3 Section 5 Standard Methods e 260 230 ratio of sample absorbance at 260 nm 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 r
110. n reconnect the USB cable At this point you may or may not see the Found New Hardware Wizard If the wizard appears follow the prompts for automatic installation of the software 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 Note If the Found New Hardware Wizard appears installation will require two cycles through the Wizard once for the spectrometer and once for the peripheral control devices that are internal to the instrument 8 1 hound Mow Hardearo Wirard Dound Now Hardware Wizard Welcome to the Found Now Hardware Wizard finder ml seach for cumani and updated software by habra det jae compas oa lhe hidro risi LO of or Ea Valarie Ll pclae Vai tbe eatin podar piiriin Harap WED Dice A T a cu Tha vnam helps you natal paaa for Di La Mirador rr bo Wiraka Ueta lo sh ha idltrame Ol pow hedeare cane eh an lla dro OD i fa Mappy dick i Cen ther bens only Ter noes and grey ime connect a devane Vala do aca rd a iad bo de Mo pot rar ral E thes ibi atria ally ac cramer 2 Iralak hom alist of ppscihc location Lhenice Intro Page Windows XP SP2 Other Windows Operating Systems 6 To access the Add Hardware Wizard for Windows 7 systems highlight the problem driver and select the Add legacy hardware feature File Action View Help SE Update Driver Software 15 c Disable est3 a ji Uninstall mputer Ek d
111. n running a routine series of standards Selecting No enables the user to enter new concentrations values for standards 1 7 The reference should remain set at 0 00 The Standards menu drop down may also be used to load a previously saved curve generate a new standard curve or view the current standard curve File Edit Configuration ENTERO Help Load from file Measure Blank View or Measure Standards Show Report Follow the steps below to either generate or modify the curve as needed e Enter the concentration for each standard The user may either click on the Active Inactive box to the left of each standard or double click any where in the row of a particular standard to bring up the Edit Standard dialog box to enter in the concentrations for each standard This box is also used to delete a single absorbance replicate value or reset the entire standard liinda ill Edit one standard Standard Standard 5 mg ml 2 000 Average absorbance 0 151 Abs Replicates esjs Cae daria A Delete AEI Selected WE ae me electe 0151 id N EE 0 153 a Standard i El i v Alternatively previously saved standard curves and standard curve concentration series may be loaded using the Standards menu bar drop down options e Measure Standards Up to 5 replicates of each standard can be measured The software will not allow measurement of samples until a minimum of either a reference and 1 standard or 2 standards
112. n the appendix for more details on this calculation e nm 1 and nm 1 abs current values of the user selectable wavelength cursor and corresponding absorbance value for a 1 mm pathlength The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e Dye 1 user selected dye e Dye 2 user selected dye e Dye 1 or 2 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 e Concentration ng ul sample concentration based on the absorbance at 260 nm and the selected analysis constant The calculated concentration is displayed in the units selected via the units drop down box The calculated concentration is displayed in the units selected via the units drop down box The units will default to ng ul each time the software is opened See the Concentration Calculation Beer s Law in the appendix for more details on this calculation e Show Report formatted for 200 samples although the buffer size can be modified Oligo Calculator The Oligo Calculator enables the user to type in a specific sequence and choose parameters associated with the oligo sequence When the Oligo Calc sample type option is first selected the following box will appear 9 10 Oligo Property Calculator x Oligo Seq AAAT
113. near zero 5 Wipe the blank from all upper and lower measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum varies no more than 0 005 A 1 mm path from the baseline Note The Nucleic Acid A280 and Proteins amp Labels modules display the absorbance values normalized to a 10 mm path so the effective variance should be 0 05 abs from the baseline 6 Reload the sample ID list before measuring samples if necessary by using the Configuration drop down box See Blanking and Absorbance Calculations in the appendix for more information on blanking and absorbance calculations 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 activated by depressing the F1 key or clicking the Measure button The Sample Position Illuminator allows the user to visualize the row of a standard 96 well micro titer plate that is to be sampled for measurement and corresponds to the sample status color coded guide on the screen See the section on Sample ID file for more information about the Sample Position Illuminator The entire measurement cycle takes approximately 20 seconds less time if fewer than 8 positions are used Re blank F3 The Re blankin
114. ng results that seem inaccurate or not reproducible it could be the result of sample or aliquot non homogeneity or liquid column breakage It may be helpful to try the following to ensure representative results e Make sure the sample surfaces are clean before starting the software module A dirty sample pedestal on startup can cause erroneous absorbance readings even negative values and signal saturation It is always a good practice to clean the sample surfaces with de ionized water to remove any dried sample that might be present Note Do not use a squirt bottle to apply de ionized water Use a 1 5 2 ul sample size Inaccurate 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 Proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form properly 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 See Column Breakage in this section for further details Heat DNA samples to 55 C and vortex before measurement Due to the small volumes required by the NanoDrop 8000 it is extremely important to ensure that the sample being
115. ngul Sempe D Lydvlys Lot h rma 122 Deta OEA Dyn 2 pmol Z 33 83 Dive 2 EE Acin m H LDP Sapte ti 1 ye dal 0357 Dyni poll 7145 ng ul Semple ID CREA Mo AN rta DIA Dye 2 aba 06 Dye 2 pobla 2554 33 76 e o _ _ i bae m it 1 EP simple 1 DyeTabs UZ DT poll 2 43 aah Sample lD Laie Mo AA mias 010 Dpedabe OE Dyno 2559 33 84 2 e r Acton im 4 1 re Sample 1 Dyn dass 0354 Dyn pmol 7457 ngul Sample ID CPVC bo A rd O13 pe 2 ba DGH Dye 2 pol 257 33 99 Active m 1 CE Semple 1 Qyetebs 0351 Dyel padi 217 ngul Sample ID Caes I AAN orm abe OG Deza UEN Dye demo 25 50 339 54 ai m 4 HP Semele 1 Dyn tabs O25 Dyn peoa 2247 ng ul Semple OVCA NE p rh mmia 0173 ps Pots 0653 Coe pb 2534 33 72 2 0 0 CNSA O46 sR e Sample Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 for RNA ssDNA 33 for single stranded DNA or Other for other nucleic acids The default is ssDNA 33 If Other is selected the user can select an analysis constant between 15 150 When navigating amongst the three 3 general sample types within the MicroArray module the last value of the constant entered within the Constant Sample Type will be retained See Concentration Calculation Beer s Law i
116. ns amp Labels Protein A280 General Settings Reports Data Exporting Nucleic Acids UV Vis Sample ID required L OFF Automatic Column Advance C OFF Prompt User to Close Data Viewer C OFF Data Exporting In addition to the primary data storage of archive files at c ND 8000 Data users may elect to export their data to an additional location This option can be chosen under the Data Exporting tab by selecting the Automatic Data Export box and then choosing the file path by clicking on the file folder icon under Data Export Folder The archived files may be opened with Excel by using the right click option of the mouse to open the nd8 file Note It is important to save the file with a new name before making changes with Excel The Export Data by Row option is applicable to the 8 sample operation mode only When selected data will be sorted in reports by plate well rows A1 to A12 then B1 to B12 rather than by columns A1 to H1 then A2 to H2 User Preferences File Help User Default Microarray Proteins amp Labels Protein 4280 General Settings Reports Data Exporting Nucleic Acids UV Vis To export NanoDrop 8000 data automatically after each measurement check the Automatic Data Export box select a Data Export Folder Optinally configure custom export data format for one or more test modules by first selecting a module and then clicking on the Configure Data Export for Module
117. ns for each method type and save multiple customized formats 7 4 e AO 0G Corte Viewer Ele Configursica Dea Pepati Hala Huciare dad 3443007 305 Pe L E Fists 9 Poport Testhypa Rapai Hama Fenosa Fill Moda igni G00rgrul_ 43 eina Defeuh 242007 TAJAM 12159 BID rigu a nul Dabah Wes 11 43 454 94 1 11503 aS GOD mul Fe eingil Duleull 3232007 TAZAM 12038 BUD esil E trata A AE 1 42 120 El mgul Bing Beth V2W2007 MATAM zz bieru Be bina Deteul 023 2007 TAE AM 11 641 Start Report Recording All data is automatically archived The user can log measurement results in an active report table as the data is accumulating by using the Start Report Recording feature in the acquisition module The default setting has the Recording feature activated for all modules If Start Report is displayed the accumulating data will still be archived but will not be shown in the active report File Edit Configuration Standards Help Show Report Selecting this button will bring up the Report page Hitting the Exit button at the top right will exit back to the acquisition module The data will continue to populate the report after exiting HD 0000 Data Viewar vi File Configuration Osia Fiepons Help Plote Report Treat hypi Pae Well Sempe Dae Time maful Iv E l Save 26k Frnt Soe and Frit o H All
118. o the A2 to H2 positions etc Each set of 96 samples are considered to be a plate Partial plates are allowable 4 2 Section 4 Sample ID Entry Options Define Sample ID File Format Sample ID File Enter the Settings for Format Sample ID Files If Use Replicates CAND 8000 Data Plate files Example t e Column is checked you must enter the Sample ID File column that holds the of Replicates If left unchecked of Replicates will be 1 for all samples in the Sample ID File pr Oa a Column 1 Column 2 Column 3 La gt EE O EE A A E Sl Samet 3 o o To Data Series Type _In Columns 41 81 Bae o pp 7 Enea J9 gt 2 2 E Sample E A IEA E Do oS Samples 3 Pp Cd of Header Rows 1 Sample 6 3 Sample a E T Sanle 3 E a eee NS Same ST O Si i a El Sane 0 ooo OOOO o oo conin ae Samet 3 gt Sh v Sample to Well Position Assignments ow L_Sempe hs l 2 3 4 5 6 E 8 9 10 11 112 Sample 63 Sample 90 Sample 3 Sample 92 Sample 93 Sample 94 Sample 95 Sample 56 Sample 80 Sample 95 TTT Selecting Continue after defining the format will load the plate file If more than 96 sample IDs are in a list the following pop up window will appear Select Plate Set amp Confirm Sample Loading The selected Sample ID file has more than 96 samples Selectthe desired Plate Set from the file Plate Set 1 al Sample ID File CAND 8000 Data Plate
119. off using the UV Vis tab in the Users Preferences module e nm 1 abs1 and nm 2 abs2 current values of the user selectable wavelength cursors and corresponding absorbencies for a 1 mm pathlength The wavelengths can be set by using the up down arrows or typing in the desired wavelength The default wavelengths are 300 nm and 700 nm e Normalize 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 400 700 nm e Show Report formatted for 200 samples although the buffer size can be modified 9 13 Section 5 Standard Methods Proteins amp Labels This software module can be used to determine protein concentration 280 nm 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 ten 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 8000 software using the Dye Chromophore Editor button found under the Tools amp Configuration button The NanoDrop 8000 Spectrophotometer measures the absorbance of up to 2 fluorescent dyes allowing detection at dye concentrations as low as 0 2 uM The respective absorbance wavelength extinction coefficient and 280 nm c
120. on 5 Standard Methods Unique Screen Features T hraddord A A Eo Edhi E Conhqurabon Standards Hein a j Measure l Diank Reblank Eocording Show Repon Luo Default Date Time 210 2000 11 01 AM Exit Pisto ILI Areciticand Measurcmmand corales a AA A Srandords L_AllActve Oniot nm 545 gt Lins ugral r T _ _ __ _ _ A _ _ _ _ Morelo Slancaach Nalid Acre t 1 Al Sample H 1 rm abe OOH Amo Um I ura A ___AA2 _ Sanpla ID i AG 0 560 1 Active 4 1 HI sample 1 mabe OTE AAD 01003 ug ml Sampe 10 i A595 0 076 an Actes n 1 CI Sample 1 mmia LU ASO W011 uafal Errana 10 _ AS 0 486 2 Artin El 1 DOI Sample ft 1 Tabs anra Amni 0008 O sye ri T ug ml sigue ICH a ARO DOTA 403 3 Ate tt 1 El Samp LEE sa um _ I sopi 1 mabe LUZ Aero WOT vail Sample ID ee ASS U 527 8 1 1 12 Actin uj 1 Fl Sepe 1 tabs 0073 Aman 0008 O is ug ml Saapke D i ARA aora 400 6 Active 1 GI Sane ii i 3 ti L mapie i rev tebe O00 APSO 400206 ug ml Sapha 10 A 06 ha Action 1 HI Sample 1 mmia MOTA APA Oe ug ml Sample ID ti ARA anA 467 6 ZU CPE a e A595 nm protein dye complex s absorbance at 595 nm at the 1mm pathlength e nm 1 and nm
121. on or delete replicates Standard ug ml AveAbs Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 A Rerne 0000 0000 oon oo oom 0001 0000 250 ome omo 0017 ome oma 003 Standard2 2500 004 004 004 004 004 0947 Standard 5000 0081 0088 0080 0082 oom 0882 Standard 4 Standards 1000 0174 0170 0173 017 0175 0175 a a IS T T T To o Standard 6 ES Standard YT OA Standard Curve Absorbance Spectra SEG TH TE GO E 0 0 1 1 i 1 fae oman 1 1 1 gt 1 1 550 560 580 600 620 640 660 680 700 720 740 750 Wavelength e Measure Samples Once a minimum standard curve has been established the standard curve indicator will change from gray invalid to green valid allowing the user to start measuring samples The designation of Valid only indicates that the minimum requirement for a 2 point curve has been met Sample concentrations are 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 Pierce 660nm Standard Curves File Standards Help Default 9 9 2008 4 02 PM Return to Sample Acquisition 2 0 0 C0158 0 30 28 Units ug ml Measurements Table Double Click on any row to change the concentra
122. orrections will be automatically utilized for measurement and concentration calculation The default setting is Cy3 In addition to the fluorescent dyes available from the drop down menu an option on the main acquisition page entitled None is also available Selecting None disables the respective calculations amp numeric displays corresponding to a 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 T M cm nm g Mol 260 nm 280 nm factor factor Insert 07 TSE 550 0000 000 000 05 250 650 o008 0 000 000 Alexa Fluor 488 710E 4 495 000 0 000 0 00 Alexa Fluor 546 1 04E 5 556 oo0E 0 ooo ooo Seemed Alexa Fluor 555 T505 555 Oo0E 000 ooo Selected Alexa Fluor 504 730674 630 oo08s0 ooo 0 00 Alexa Fluor 64 230Es5 650 oo0e 0 ooo 000 Alexa Fluor 60 T325 oss oo0es0 ooo 000 035 T505 581 oooe 0 ooo aoa Os res0e s 675 oooe 0 ooo 000 Test raaoe o 220 ooe ooo 0 00 a Note predefined dyes are indicated with a diamond and cannot be modified Sample Volume Requirements Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter the surface tension resulting in difficulty forming and or maintaining adequate columns for measurement The column formation issue can be ove
123. quirement The presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter the surface tension resulting in difficulty forming and or maintaining adequate columns for measurement The column formation issue 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not from properly 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 Using the regular Bradford assay the concentration range of detection is 1
124. r detergents in reagents can significantly alter the surface tension resulting in difficulty forming and or maintaining adequate columns for measurement The column formation issue 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 Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form properly 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
125. r 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 e Low Detector Bias This occurs when the software has identified a problem with the detector Contact your local distributor or Technical Support if you encounter this error 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 100 MB of free hard disk space 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
126. ral 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 10 1 Section 10 Appendices Dye Chromophore List Editor Dye Chromophore List Name T M cm nm g Mol 260 nm 280 nm factor factor 07 oes 550 ooe ooo oo Below 07 2508 5 650 000 0 000 000 Alexa Fluor 488 710E 4 495 0 00 0 000 000 Alexa Fluor 546 1 04E 5 556 oo0E 0 ooo ooo Seemed Alexa Fluor 555 T505 555 0owes0 ooo 000 ES Alexa Fluor 50 raver 590 ooo 000 900 Alexa Fluor 64 2308 5 650 oove 0 ooo ooo Alexa Fluor 660 T325 663 oove o ooo o0 0 35 Tso sai ooe ooo 000 05 2508 5 675 000E ooo 00 Tes Fosoe 0 220 ooo ooo omo AAA A Note predefined dyes are indicated with a diamond and cannot be modified Nucleic Acids For nucleic acid quantification the Beer Lambert equation is modified to use an extinction coefficient with units of ng cm ml Using this extinction coefficient gives a manipulated equation c A e b Where c is the nucleic acid concentration in ng microliter A is the absorbance in AU e is the wavelength dependent extinction coefficient in ng cm microliter and b is the path length in cm The generally accepted extinction coefficients for nucleic acids are e Double stranded DNA 50 ng cm ul e Single stranded DNA 33 ng cm ul e RNA 40 ng cm ul For
127. rce 660 nm assay is run Although curves can be saved and reloaded in the NanoDrop 8000 software it is recommended that the user follow manufacturers guidelines and generate new standard curves if appropriate Both single and multi point standard curve generation is incorporated into the software A standard curve can be developed using a reference assay reagent only no protein and a single replicate of one standard There is no set order in which standards must be run The multi point standard curve generator allows a maximum of 5 replicates for each of 7 different standards The following box will appear after the module initialization is complete Choose Standards Source Standards are required Choose the Standard Curve source Load Standard New Standards Curve Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Measurements Table Dibulblo CEHE Gn Griy ni aoe tht TEN r d ls ricana Sandard mall don ARS Ab Abs 4 4 1 0UO 1 000 i 3000 4 500 Clicking on the Yes button will allow the user to import just the Standard series without the respective measured values See image on the right above This option is very useful when running a routine series of standards Active E A Reterence Do you want to load the concentrations and curve type for
128. rcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not from properly If this occurs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found at on our website 5 14 Section 5 Standard Methods Measurement Concentration Range The NanoDrop 8000 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 all Coa pelveton Upper minimum 5 replicates yp Limit SD mg ml C
129. rds Help Prim Pega Lies upei Mossura mk Measurements Takibi B titi ACAD 2 H Stenderd y Sia 3 Acie E A IEA Acija o D Sada ACH O E Standarnd 4 ripa o fF Giandand 700 CPE AO Double Click on ay m0 to change the concentration or Galete replicalas be L USE a i i oar i 000 07110 4000 Aono iz Standard Curve Absorbance S Spoctra 03 Eiandard ie Type Interpolation 2000 0 40000 5000 60000 70000 00005 ugiml Exiting the Bradford Module It is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module 9 28 Regular Bradford curve using a 50 1 reagent to sample volume covers the range of 100 8000 ug ml Note the linear range is 100 1000 ug ml A Bradford assay using a 1 1 reagent to sample volume covers an approximate range of 15 100 ug ml Section 5 Standard Methods 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 Sample Volume Requirement The presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter the surface tension resulting in difficulty forming
130. ription of this feature and the following message will appear when the user attempts to make a sample measurement e Auto Advance Columns If selected there is a 5 second delay before the displayed spectra clear and the status code and Sample Position Illuminator see below for description of these features advance to the next column for sampling This gives the user the option of canceling auto advance for that column Auto advance will restart when the next column is measured This feature can be set as a default function under the User Preferences application Auto Column Advance Automatic column advance will occur in 4 6 seconds Advance Coluimn Now Cancel Note All data is automatically archived and is accessible though the Data Viewer e Prompt Close Data Viewer If selected the user will be given the option of closing the Data Viewer when exiting the software module e Measurement Limits Allows the user to set minimum and maximum concentration limits for samples to be measured Additional information about setting measurement limits is included in each application module section 4 4 Section 4 Sample ID Entry Options Enter Measurement Limits Enter the Minimum and Maximum Concentration Sample measurements that fall outside this range will be highlighted in displays A Min Concentration 10 00 za Max Concentration 3000 v Continue Cancel e Show Plate Summary Allo
131. rives Scan for hardware changes play adapters Add legacy hardware D CD ROM drives y disk driv Properties RE Dppy drive controllers Help man Interface Devices j 50 ATA ATAPI controllers ES Disk Management Keyboards Sy Services and Applications A Mice and other pointing devices E Monitors amp Network adapters a fs Other devices jy PCT Serial Port PCI Simple Communications Controller ite Thermo Scientific NanoDrop Product YY Ports COM amp LPT 7 Ignore the instructions on the first screen regarding the installation screen and click Next 8 Choose the Advanced selection and then select Next 9 Scroll down through the devices listed alphabetically to Thermo Scientific NanoDrop highlight and double click Select Next 10 Select Have Disk to bring up Install from Disk window 11 Select the Browse button and navigate to the correct file location from which the drivers should install For most computers this string will be as follows Local Disk C Program Files x86 Thermo NanoDrop 2000 ndusb32 or 64 12 Click Next to start the installation process When complete a code 10 This device cannot start message will appear This is expected and indicates that both the original problem device and the newly installed device driver are installed Click Finish 13 navigate back to the Device Manager The two NanoDrop devices may be located in several places In the example below you would
132. rop down menu minimum and maximum concentration limits can be set for protein mg ml measured with this method at 660 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 29 Section 5 Standard Methods Unique Screen Features File Ed Configuration Standards Help Measure Blank Rp blani Recording Show Report User Detasult Deto Tome S008 4 35 PM Piate ID Meosuremem complete ii All Active On O mn B0 S ugm Standards View Update Standards E Active O 1 Sample Nabe gt z naan ug ml Sample ID Pierce 660 a a 000 Actor a O ug ml Sample iD Pinson BAD A 67 45 Awel mi 1 5 rm 1 abe ug ml Sample ID Fierce E5U 65 289 1 Actes BY O ug ml Sample ID Pierce 650 t E 541 Y Aawe al 1 5 rm1 ats ug ml Sample iD Pisses FED 700 5 Active 1 Semple ID Pierce G60 Ks A 017 S ee eOOOOO0000 9600000000000 La ug ml 000000000000 ell Pires Fen A880 023 NaN 9 09000000O000O0O El 31000 000000000 EE ug ml 0600000000000 e o Nias 20
133. rtmmand comple All Active Onjom ami 350 nme 700 4 Acre t 1 My Al Sample 1 rm Tete OM nm Z abe UUU Sree unikna T YN a aawa a 1 VAN Cl Sempet 1 ren Tate 03 sn Z abr 0 001 Sample D aan d o A dr H 1 AA OI Sempet 1 rmiahs AFA nm2abs 1001 ine IA Sample iD unknown 4 Moneda fe m r z 7 haa Al a N A Simple 1 1 rel ados OF nm abe W002 Gamme O A E 1 1 12 a OO OOOO OOOO semelb unknowns Activ 1 VA i Sample tt 1 mide 072 rem2aba 0000 1800000000000 A Be 1 600000000000 same 10 ikr WMODODOCOCDODO Ca aes z En Sacra H 00000000 e 00000000000 sao ra MN Sample H i nmila 07 nm ebi 0 001 Hil a Pe F m 4l 1 Acton 1 ae BT Sample 1 rmi aha OFAR nm tabs C006 Sample ID unknown 2 fe 4 CUO CPE Oa Automatic Path Selection If selected the software will automatically switch from using the 1 0 mm pathlength to the 0 2 mm path when the absorbance value first reaches 1 25 for EITHER of the two wavelengths indicated with the two cursors Note It is important to pre select a cursor position for the wavelength of interest before the measurement is taken If the cursors are set for wavelengths with minimal absorbance the 0 2 mm pathlength will not be utilized Note When the 0 2 mm pathlength is utilized the data will be archived and displayed normalized to the 1 0 mm pathlength The feature can be turned
134. s See image on the right above This option is very useful when running a routine series of standards Selecting No enables the user to enter new concentrations values for standards 1 7 The reference should remain set at 0 00 The Standards menu drop down may also be used to load a previously saved curve generate a new standard curve or view the current standard curve ET ards Help Load from file View or Measure Standards Then follow the steps below to either generate or modify the curve as needed e Enter the concentration for each standard The user may either click on the Active Inactive box to the left of each standard or double click any where in the row of a particular standard to bring up the Edit Standard dialog box to enter in the concentrations for each standard This box is also used to delete a single absorbance replicate value or reset the entire standard Edit one standard Standard Standard 4 mg ml 1 000 Average absorbance 0 249 Abs Replicates Delete zs arene 0 250 aS 0 254 0 25 Reset v Alternatively previously saved standard curves and standard curve concentration series may be loaded using the Standards menu bar drop down options e Measure Standards Up to 5 replicates of each standard will be displayed The software will not allow measurement of samples until a minimum of either a reference and 1 standard or 2 standards are measured Polynomial curve fitting requires mor
135. say is run Although curves can be saved and reloaded in the NanoDrop 8000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay 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 displays a maximum of 5 replicates for each of 7 different standards There is no set order in which standards must be run The following box will appear after the module initialization is complete 9 22 Section 5 Standard Methods Choose Standards Source Standards are required Choose the Standard Curve source Load Standard New Standards Curve Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Do you wantto load the concentrations and curve type for generating a standard curve from a previously saved standard curve file Active E E Standard 4 pete B C Senaera E J 3 000 PO Arie o hatine E hacir E Clicking on the Yes button will allow the user to import just the Standard series without the respective measured values See image on the right above This option is very useful whe
136. sh aliquot of CF 1 1 5 ul Allowing the CF 1 to remain on the pedestals even for a short period of time eg 15 20 seconds will allow the CF 1 to concentrate and may result in higher than expected absorbance values Lower the arm and click the Measure button After the measurement use a lab wipe to remove the CF 1 from the both upper and lower pedestals change tips and repeat steps 11 to 12 for a total of 5 sets of measurements Calibration Check Results After the 5 measurement the results will be displayed on screen When the instrument passes the check procedure a pop up box will indicate that the instrument works within specifications and the results will be saved as both a JPG and a IXT file in the ND 8000 Data Calib check folder on the local drive If the calibration check results in a failure or conditional pass it is recommended that you first Save and Review the data and then review each series of data points looking at trends associated with individual positions The tolerances for the calibration check are available under the Help drop down menu The actual values utilized are dependant on the CF 1 lot target value Refer to the following troubleshooting guide If the Calibration Check warning box appears 7 10 Troubleshooting Root Causes Resolutions minutes single set of aliquots measurement single set of pipette tips measurement e f more than 10 seconds does High elapse wipe away the samples and absorb
137. sition page allows the user to define what units to utilize when displaying and archiving calculated concentrations Note The data will be archived using the units chosen at the time of the measurement Although the concentrations may change in the display as one selects different units from the drop down the archived values will not change From the drop down box select Edit List to bring up the following pop up box Units List Editor Current Units List New Units ea mM mM ug ml uM ug l Scaling from g L 0 00E 0 4 nhi mg l mojm ng nl Update Selected ng ul Ed Units with a diamond symbol are protected amp cannot be deleted Select and drag any unitto re order the units list Weight Based Molarity Based v NOTE the Mol Wt must be known for the sample type being measured to convert between weight and Molarity based units Enter in the name for the desired new units and include the appropriate weight based or molarity M scaling For those modules methods for which a molecular weight value can be entered the units selection accommodate both weight and molarity based scaling options A molecular weight is required to convert between weight and molarity Sample ID The Sample ID is highlighted for overtyping or barcode scanning The user may input a sample ID that will be used to identify the measurement in a report print and in the archived data file The sample ID entry is key
138. stals 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 with 5 ul of dH20 to remove any residual HCl Pedestal 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 no more black residue To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up Cleaning of Light Source Window The Xenon flash lamp window must be kept free of debris and obstruction The window may be cleaned with a water dampened laboratory wipe Note Do not use organic solvents such as acetone to clean the window Decontamination of Measurement Pedestals If
139. sumption is 3 W and the flashlamp 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 Note It is recommended that the instrument not be positioned in a way that makes it difficult to unplug the power supply from the unit or the wall 2 2 Section 2 Initial Set up Registering Your Instrument Please register your product We periodically update our software and add new features free of charge To download the latest version of software from our website you must register your instrument All information supplied is completely confidential You can register your instrument on our website Lock Attachment Port The NanoDrop 8000 is equipped with a lock slot that enables use of a standard locking cable typically used to secure a laptop PC 2 3 Section 3 General Operation 3 General Operation 1 Position the instrument at an angle for optimal use of the pipette guide With the sampling arm open position the pipettor using the guide as shown above Dispense the samples onto the lower measurement pedestal ensuring the samples touch off contact the lower pedestal If the tips splay or skew when touching the pedestal surfaces please use an alternative brand or style with a more rigid tip structure Optimal position for right handed Loading samples when operating Loading samples on position A pipetting onto the pedestals within the 8
140. t Date Time 9 9 2008 2 46 PM ea Plate ID Make new BLANK Measurement nC Sample Type DRA All Active On Off nm1 260 Units ngul Blank F2 Before making a sample measurement a blank must be measured and stored All eight positions are blanked with each blanking command when using the 8 sample mode Only position A is blanked for the single position mode Note When using the 8 Sample mode the software initiates each blank and measurement cycle on the first position to be read The user will therefore hear one less position increment than expected After making an initial blank measurement a straight line will appear on the individual graphs Subsequent blanks will clear any sample spectrum and again display straight baselines 3 3 Section 3 General Operation Blanking Cycle For the most consistent results it is best to begin any measurement session with a blanking cycle 1 Open the application software module 2 Use an 8 channel pipettor to load an aliquot of the blank the same buffer or solvent the unknown samples are in onto each of the lower measurement pedestals and then lower the sampling arm to the down position 3 Click on the Blank button When the measurement is complete wipe the buffer from all pedestals 4 Select All Active On and analyze a fresh aliquot of the blanking solution on all pedestals using the Measure button F1 The result should be 8 spectra with relatively flat baselines
141. t format as an nr8 file for retrieval and future use To designate a saved report format as the default format exit to the Main Menu choose Users Preferences and click Reports Use the Select Default Report Format to see the list of saved formats available for the specific method type e Load Report Format Allows saved report formats to be loaded either before or after data is imported e 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 e Save Report and Load Report There are several options for this feature as seen in the following window Save Report As Choose how to save the report Choose this option to be able to reload the Aula report into the Data Viewer at a later date Choose this option to export a tab deliminited textfile ofthe displayed Report Table suitable for import into Excel Export Report Table Only Choose this option to export a tab deliminited text file of the displayed Report amp Standards Tables suitable for import into Excel Export Report amp Standards Tables 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 confi
142. t 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 for assistance 8 4 Error Code 8 Error Code 8 Error reading or writing to file This might be caused by 1 The current indows account does not have Read and Write priveleges to the folder CAND 5000 Data and all of its subtalders Contact your PC administrator to give all users Read and Write access to these folders e The log files have been removed from the folder CANO SOO0 Datailog tiles Replace the log files ifthey have been moved Ifthe log files can not be located reinstall this sofware 3 The log files described above are setto read only Check the properties on each log file and ensure thatthe Read only box is unchecked This error code is most likely to occur if the user does not have read and write access to the folder c ND 8000 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 Error Code 8013 Error Code 8013 ND 8000 Peripheral Control Device not found The software installation process installs two USB drivers The above error message indicates that the motor UBS driver was not properly installed Follow the instructions given above for the Instrument Not Found error When att
143. the upper and lower measurement pedestals by extraction with a pipette Single vs 8 Sample Modes The NanoDrop 8000 may be used in either a Single Sample or an 8 Sample mode The 8 sample mode will appear as the default each time the software is opened NanoDrop 8000 V2 1 0 File Help User Default v Standard Methods User Methods Tools amp Configuration Single Sample 8 Sample Proteins amp Nucleic Acid Labels Protein BCA Protein A260 Protein MicroArray Bradford Protein UV Vis Lowry Cell Cultures Protein Pierce 660 nm pH HEEE Functions Common to Single and 8 Sample Modes Module Startup When a software module is opened the first message seen will indicate that the instrument motors are initializing A second message will then appear with instructions to load water aliquots to initialize the spectrometer For best results ensure measurement pedestal surfaces are clean and load 2 ul of water onto each lower measurement pedestal Lower the arm and click OK The message Please wait Initializing Spectrometer will then appear When this message disappears the instrument will be ready for use All data taken will automatically be logged in the appropriate archive file Note It is only necessary to load water onto pedestal A when running the single sample mode Nucleic Acid Eile Edit Configuration Help Measure Blank Re blank Show Report User Defaul
144. the Dye Chromophore Editor button found in the main menu Dyes can be selected using the scroll arrows or by highlighting the Dye box The respective absorbance wavelength extinction coefficient and 260 nm and 280 nm corrections will be automatically utilized for measurement and concentration calculation The default setting for Dye 1 is Cy3 and Dye 2 is Cy5 See User Preferences under the Tools amp Configuration main page tab to designate alternative dyes as the defaults 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 g Mol 260 nm 280 nm 4 factor factor e reves 650 ooe ooo ooo Below 0 00 07 2508 5 650 ooo 0 00 le Alexa Fluor 488 710E 4 495 000 0 000 0 00 Alexa Fluor 546 1 04E 5 556 o 00e 0 ooo ooo L Bee Alexa Fluor 555 SOE 555 ooe ooo omo ES o Alexa luar 59 730594 580 ooe ooo foo Alexa Fluor 64 2308 5 650 000E ooo o0 Alexa Fluor 660 T325 663 o00E 0 ooo o0 0 35 Tso 561 ooe ooo 000 075 2s0e 5 675 oove 0 ooo 000 Test roove o 220 ooo ooo ooo 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 fluor
145. the NanoDrop 8000 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 8000 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 Solvent Compatibility The NanoDrop 8000 Spectrophotometer is compatible with most solvents typically used in life science laboratories These include methanol ethanol n propanol isopropanol butanol acetone ether chloroform carbon tetrachloride DMSO DMF acetonitrile THF toluene hexane benzene sodium hydroxide sodium hypochlorite bleach dilute HCI dilute HNO3 dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable 10 2
146. tion or delete replicates l Standard ug ml AveAbs Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 Measure ive M _A Peference 0000 0000 oom 0001 0007 ooo 0000 i Standard A Pierce 660 nm Standard 004 z Standard3 5001 assay using a Standard 4 15 4 Standards 1 reagent to Standards gt gt pp 7 sample volume sendera __ _ _ covers an l approximate 0 2 range of 50 2000 Standard Curve Type Linear ug ml D 1 Pesquared 0 9929 o 2 Ll 0 0 0 1 00 1000 2000 3000 4000 5000 600 0 7000 8000 9000 1000 0 ug ml Exiting the Pierce 660 nm Module It is recommended that you process all of the unknowns to be assayed before exiting the software module 9 32 Section 5 Standard Methods 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 between the optical systems of the numerous spectrophotometer designs Note The most distinct difference between the NanoDrop 8000 Spectrophotometer absorbance values for cell microbial 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 i
147. tometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Cell Suspension Concentrations Due to its shorter pathlength the NanoDrop 8000 can measure absorbances that are 10 fold higher than those measurable on a standard cuvette spectrophotometer It is important to note that the absorbance reported is that for the 1 mm path and will be approximately 10 fold lower than absorbance values reported when samples are measured with a traditional cuvette based spectrophotometer Since the entire spectrum is displayed diluted samples exhibiting very low Absorbance at 600 nm can be monitored at lower wavelengths for example 300 nm Sample Homogeneity The user must be sure to homogeneously suspend the cells when sampling for absorbance measurements and read the sample immediately to avoid significant cell settling Vigorous mixing may be required particularly when measuring concentrated samples Decontamination of Measurement Pedestals If decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach solution freshly prepared can be used to ensure that 9 34 Section 5 Standard Methods 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 Comp
148. ts be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form properly 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 8000 Spectrophotometer will accurately measure protein samples up to 100 mg ml BSA without dilution To do this the instrument detects the high concentration and automatically utilizes the 0 2 mm pathlength to calculate the absorbance The table below lists the concentration range and typical reproducibility for purified BSA measurements on the NanoDrop 8000 Approx Typical Reproducibility Sample Type peta HON Upper minimum 96 replicates Limit SD mg ml CV sample range 0 15 5 mg ml 0 15 mg ml Purified BSA 0 15 mg ml Ponga sample range gt 5 mg ml 2 5 By selecting Measurement Limits from the
149. uration at set up If measurement limits have been defined wells that are out of the defined range will flash on the Sample Position Illuminator immediately after measurement These positions will continue flashing until measurement of the current plate sample set is complete the results have been reviewed and the Plate Results Summary window is closed Plate Review The Plate Results Summary window will automatically appear if the auto advance column feature has been selected and measurement of the plate sample set is complete Selecting Show Plate Summary from the configuration drop down menu will also open this window From this window sample wells of interest can be marked for repeat If measurement limits were defined sample measurements that are 4 5 Section 4 Sample ID Entry Options outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator and the concentrations will appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing Plate Results Summary File Plate ID 425 ng ul Plate Results nq ul 1 2 3 4 5 6 7 8 9 10 11 12 458 2 EN 4596 460 2 4603 4600 4603 4589 4580 4595 4586 458 8 4585 4609 4609 461 2 4596 4606 4659 461 0 4598 461 2 461 1 4614 4569 4588 4565 4577 4569 4578 4589 4583 4582 458 4 461 6 457 4590 4592 4587 4586 4590 4
150. ured 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 8000 where it is recommended that water be used as the blank The following box will appear after the module initialization is complete Choose Standards Source Lisad Standard Min andad Curva _Mensuramana 5 30 Section 5 Standard Methods The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Do you want to load the concentrations and curve type for generating a standard curve from a previously saved standard curve file G Exarlara E recite va FH Standard 7 Clicking on the Yes button will allow the user to import just the Standard series without the respective measured values See image on the right above This option is very useful when running a routine series of standards Selecting No enables the user to enter new concentrations values for standards 1 7 The reference should remain set at 0 00 The Enter Standards Manually button allows the user to type in a predefined set of concentration and absorbance values that are supplied by the reagent manufacturer The Standards menu drop down may also be used to load a previously saved curve gener
151. v Fuk Hal 7 569 Action H BI os 3 j Sample H Dyr Taba 4276 Delw 285 se te Sample 10 DAA cud il pm iabe 4923 Cpe Pole MaN Dye 2ukl Hah FA Actor 1 CI Semel 2 Dys Tate 4 327 Diu WE Dye 1 6 gt 2 ya i Ei in 0 83 mg ml Sample Ty Bo cyt A ma 5013 Dye abe Mah Dyeu AM 7 315 Dre 2 Marne w Awn H 1 OT sap 2 Cig The 4300 Cipo Tuk SA ER Baseline Type 750 nmi na a gimi k Semple 0 BSA m3 ha ath miah AST Cpe abe MaN we Du Ha 2250 24410 Bichromate fr AE a y E Se Atrea Hi 1 5 bil SE Bb L H Semple 2 Dust 412 Dye Tull 28 64 Tal 1 1 Sample ID Far ey Le Wim 4970 Dip ake MaN iva Fudd Ha 7 238 A1000000000000 An 4 1 H Sempe t 2 Gyn dabs 4271 Dyn 1 bl 2847 majal HIBOOOCOOOC OOOO sell FAA eya Lr em dos 4984 A Dye 280 HoN 7 201 Cc Active 1 GI Gaani ii i 52 5 e 900000000000 m a DiR CBE mgl E 00000000000 tame Bans AN rem abt 4 002 Dye abe Mall Dye Zum NaN zoi F Wee ee ele Acton H 1 l HI Sampean Diy lake 433 Dyn Tab 201 mg ml i j Semple lD BSA ey l A Tohr 4785 Deza Ha Hol 7 110 600000000009 tt BS A eae 20 0 COE Oa e Sample Type The same six sample types options listed in the Protein A280 section 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 t
152. when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Measurement Concentration Range The NanoDrop 8000 Spectrophotometer will accurately measure dsDNA samples up to 3700 ng ul without dilution To do this the instrument detects the high concentration and automatically utilizes the 0 2mm pathlength to calculate the absorbance The table below lists the concentration range and typical reproducibility for nucleic acid measurements on the NanoDrop 8000 Detection Approx Typical Reproducibility Limit Upper Limit minimum 96 replicates ng ul ng ul SD ng ul CV 3700 ng ul dsDNA 95 3000 RNA sample range 2 5 100 ng ul 2 5 ng ul 2400 ssDNA sample range gt 100 ng ul 2 5 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be set for nucleic acid measurements as defined by the absorbance at 260 nm These limits cannot be set as a default and must be defined each time the application module is opened Nucleic acid measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of inter
153. ws the user to review the results of the plate at any time during the measurement session Once the entire plate has been read this feature will allow the user the option of marking samples of interest for repeat testing Sample Status Color Code The 96 well plate schematic allows the user to monitor the position of the samples being measured Black wells indicate that samples in the corresponding wells have been measured Green wells indicate which positions are currently being measured while yellow represents well positions that are expected to be measured Pre loading a Sample ID file or manually entering in sample names will activate the respective wells To select additional wells use the Configuration drop down box and select Manual Plate Set up If Sample ID Required has been selected from the configuration drop down any wells that do not have an ID assigned will appear red and a sample ID must be assigned before measurement can begin Ka EA z e 9 e e e e e e e OOO as mi a ue l A e ies 00000000 0 0 0 0 0 0 0 0 OOOO lol lOlOIDIDIOIO IOI lOIDICIOIOIO IOI QA AO Weee 010 88 O Sample Position Illuminator This lighted guide allows the user to visualize the row of a standard 96 well micro titer plate that is to be sampled for measurement The Sample Position Illuminator corresponds to the sample status color code displayed on the software screen and the pattern of illumination is determined by plate config
154. x the CF 1 Calibration Fluid by vigorously shaking ampoule and then tap upright ampoule lightly to ensure all solution is collected in the bottom portion of the ampoule Carefully break the neck of ampoule to open the CF 1 Calibration Fluid by snapping the vial away from your body using the plastic snap guard Aliquot 55 ul into each of well of the second 8 well strip Use the 200 ul volume pipettor set to 55 ul for the transfer Use a manual 8 channel pipettor to simultaneously draw up eight 1 5 ul aliquots CF 1 Tip When drawing CF 1 solution up into pipette tips 1 5 ul quickly check by visual inspection that each tip contains equal amounts in all 8 pipette tips If one or more tips is missing CF 1 or has less volume than the other tips discard the CF 1 and tips Using a new set of 8 tips draw up a fresh aliquot of CF 1 1 5 ul 7 9 12 13 14 Use the Discharge and Touch Sample Delivery method as described above to simultaneously pipette 1 5 ul of CF 1 onto the pedestal Tip Very quickly visually check that each pedestal has equal amounts of CF 1 droplets and the droplets are centered on the pedestals If one or more pedestals has a smaller volume of CF 1 or the CF 1 is not centered on the measurement pedestal wipe the CF 1 away discard the pipette tips and draw up a fresh aliquot of CF 1 1 5 ul Tip If the visual inspection takes more than a few seconds wipe the CF 1 away discard the pipette tios and draw up a fre
155. ys in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not from properly 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 Modified Lowry assay concentration range of detection is 0 20 mg ml to 8 0 mg ml BSA on the NanoDrop 8000 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be defined for protein mg ml measured with this method at 650 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also

NanoDrop 8000 Spectrophotometer V2.2 User Manual

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