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NanoDrop 8000 Spectrophotometer V2.2 User Manual

1. 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 Nradford Standard Curves filo Stondeeds Help Pint Fogo 40000 179 80000 T 0205 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 5 44 Section 5 Standard Methods Bile Standards Help _ PrimPage Units ugi Moasure Blank Dotout Ronam to Sample Acquisiton 200 CPE O01 32 NATIS IAM Double Click on any row to change the concertraton or dalete replicates agin Ave Abs Abs Abs 3 Abs 5 S600 owi 1000 omo 5 Standard b Standard 7 w or 6000 0130 Standard 5 2000 0139 Standerd
2. 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 Features include e Import folder Used to select folder where data is to be imported from Folder selection must be at the level of user or higher may not select an application or method folder within a user in this activity box 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 7 4 Section 7 Tools amp Configuration function keys will allow the user to select multiple s
3. dilute HNO3 dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable Decontamination of Measurement amp Optical Surfaces If decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach 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 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 10 3
4. 260 280 Ratio in the Troubleshooting section for more details on factors that can affect this ratio 260 230 ratio of sample absorbance at 260 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 ratio is appreciably lower this may indicate the presence of co purified contaminants Conceniration 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 Show Report formatted for 200 samples although the buffer size can be modified 5 6 Section 5 Standard Methods 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 AAATTTCCCGGQ NucleicAcid DNA Modification Weight oo 4 Phosphorylated Double stranded C Mol Weight g mol 3 645E 3 GC 50 00 Ext Coeff I rmal cm 1 280E 5 Conc Factor ng ul 28 48 of bases 12
5. OK Cancel 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 are referenced off of this zero 5 7 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 rea
6. 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 Bovine Serum Albumin reference Unknown sample protein concentrations are calculated He using the mass extinction coefficient of 6 7 at 280 nm for a 1 10 mg ml BSA solution IgG reference Unknown sample protein igG concentrations are calculated using the mass extinction coefficient of 13 7 at 280 nm for a 1 10 mg ml IgG solution 5 10 Section 5 Standard Methods Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution User entered values for molar extinction coefficient M cm and molecular weight MW in kilo Daltons for their respective protein reference Maximum value for e is 999 X 1000 and maximum a value for M W is 9999 X 1000 Mol Weight kDa Sample Type Other protein E 1 User entered mass extinction coefficient L gm cm for a 10 mg ml 1 solution of the respective reference protein Ext Coeff E 1 Ligm cm 1C 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 wavelength can be set by u
7. enter all sample names in one column and the number of replicates desired for each sample in another 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 posit
8. 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 Upper minimum 96 replicates Limit SD mg ml CV Sample Detection Type Limit i sample range 0 15 5 mg ml 0 15 a 0 15 mg ml 100 mg m mg ml sample range gt 5 mg ml 2 5 By selecting Measurement Limits from the 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 5 9 Section 5 Standard Methods Unique Screen Features Bilo Edt Conhgurahon Help Measure Blank Rebdlonk Roconing ShowRepotn user Default Date Time 9 10 2000 454M Ext Plate ID HSA Measurement complete om Sample Type
9. 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 E EA N mTonmmoow gt r JOCO OOOO O O O O O O O eo O O le e D O is D O D OD O 0 O O O O O DO sooo eooo Jeees seoa J eleielele oeeie sooo J elelelele 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
10. 045 0 046 0 046 0 047 D Standard 3 500 0 0 091 0 088 0 090 0 092 0 091 0 092 E Standard 4 750 0 0 120 0 120 0 118 0 119 0 121 0 123 F Standard 5 1000 0 174 0 170 0 173 0 175 0 175 0 175 G Standard 6 H Standard 7 Standard Curve Absorbance Spectra 0 2 Standard Curve Type L Linear p o 2 od Pe d square oo moai 0 9929 14 0 0 1000 2000 3000 4000 500 0 ug ml 6000 700 0 800 0 300 0 1000 0 Exiting the Pierce 660 nm Module It is recommended that you process all of the unknowns to be assayed before exiting the software module 5 51 A Pierce 660 nm assay using a 15 1 reagent to sample volume covers an approximate range of 50 2000 ug ml 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 e
11. 19810 U S A Telephone 302 479 7707 Fax 302 792 7155 E mail info nanodrop com www nanodrop com For International Support please contact your local distributor Microsoft Windows Windows NT and Excel are either trademarks or registered trademarks of Microsoft Corporation in the United States and or other countries Adobe and Acrobat are trademarks of Adobe Systems Incorporated All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries NanoDrop is a trademark of Thermo Fisher Scientific Revised 3 2010 Table of Contents DOVER VICW AEAEE A aaan E AEAEE EAEE E E 1 1 Instrument Description cceseececeseeeeeeseneeteneeceeenseeeneeseneeseneeeeeseneenens 1 1 Operations cache eaaa non a E N ciate ee A 1 2 SUEI AEE E E E A E A ET 1 2 WEEE Compliance esis sry tescaevin ees n iS a T dite 1 3 Patents ins hast penta evites oot ae hii ete teens See aE 1 3 2 Initial Set UP iiien anaiaren leecceesiceeveineseenuelvclecsscsedeteeteseecss 2 1 Computer Requirement c cceecceeseeeeeeeeeeeeeeseeeseaeeeeeeeeaeeeseeetsaeensaeetias 2 1 Software Installation eecceceeeeseeeeseeeeseeeeeaeeeseeeesaeeeseeessaeeeseeeseaeessaeereas 2 1 Registering Your Instrument esseceeeeseeceeeseeeeeenseneeeeseneesenseeeeestenenes 2 4 3 General Operation cssccscecceseeeeeseeeseeeseseeeeneeeeeseeeseneenenseeeeenees 3 1 Cleaning the Sample Retention System ecceceeeeeeeseeeee
12. 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 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 g Mol 260 nm 03 1 50E 5 0 00E 0 0 00 i Below OF 250E 5 D00E 0 0 00 i Alexa Fluor 488 495 0 00E 0 Alexa Fluor 546 1 04E 5 0 00E 0 0 i SBEGed Alexa Fluor 555 1 50E 5 0 00E 0 i Edit Alexa Fluor 594 7 30E 4 0 00E 0 i Alexa Fluor 647 2 39E 5 0 00E 0 Alexa Fluor 660 1 32E 5 0 00E 0 O35 1 50E 5 0 00E 0 O55 2 50E 5 0 00E 0 Test 0 00E 0 0 00E 0 Note predefined dye
13. 495 280 A 495 A 260 30 lt lt lt Available Formulas Formulas used with this Method 2 maximum Edit Formulas The final page of the wizard allows the user to name and describe the new method 6 4 Section 6 User Methods Edit method parameters Step 5 of 5 Name Method name Alexa 488 Protected Description fluorescent dye optional Back Finished l Cancel 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 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 6 5 Section 6 User Methods Edit method parameters Measurement Type Wavelengths Baseline Name Sfi Eom g Quantification Mode c FactorxA b v ie ESTEE is calculated by multiplying the absorbance A at the Analysis wavelength by the Factor and dividing by the path length Both the Analysis nm and the Factor ar
14. 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 7 1 Section 7 Tools amp Configuration E Microsoft Fxcel Nucleic Acid 2005 11 01 v3 2 ndj Read Only i fle Edt View Insert fomi Too ste Window ACTI tiep Adobe FOF 10 B 7 Dm MS gt Al amp Module 8 fomesrp TE F ES SS SS SSS See SS a Seay Module Nucleic Acid 2 ath 10 mm e 320 4 e USB2000 2 41 3 NOS E Sample ID User iO Date AJD AD W0 WVD Constant Cursor Pos Cursor abe 340 raw Measurem 6 Default a G NaN NaN o 230 0 0 Blank rl Default 007 10 242 212 1 230 0003 N Measure 6 Default 0 60 0 43 50 230 0 0 000 Measure 9 Default 7 67 427 2 50 230 3 353 0 006 Measure 10 Default 74 08 33 04 1 66 2 24 gu 20 3075 00W 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 C ND 8000 Data Default Microarray be Go Folders X Name Size Type Date Modified ND 8000 Data A S MicroArray 2007 03 21 v1 14KB ND8 File 3 21 2007 4 02 PM S Administrator E MicroArray 2007 03 23 vi 18KB
15. 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 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 8 1 Section 8 Troubleshooting a Computer Management Local ry Batteries 5 A System Tools yg Biometric f Event Viewer i Bluetooth Shared Folders 4 Computer Local Users and Groups Se Disk drives Performance Logs and Alert 2 Display adapters Device Manager DVD CD ROM drives os Storage IDE ATA ATAPI controllers Removable Storage Sj IEEE 1394 Bus host controllers Disk Defragmenter Keyboards Disk Management 75 Mice and other pointing devices fe Services and Applications b Modems Monitors NanoDrop Devices NanoDrop LED Position Indicator lt 4 ND 8000 Peripheral Control Device ND 8000 Spectrophotometer 4 B Network adapters PCMCIA adapters Ports COM amp LP
16. Cure Absorbance Spectre 03 Standard Gave Type Imerpolahon 10009 20o 000 Exiting the Bradford Module It is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module 4000 5800 0 60000 7000 00000 ugiml 5 45 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 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 recommende
17. If the instrument needs to be recalibrated contact your local distributor or Technical Support for assistance Common Technique Issues Improper Blank When individual positions appear to have very low absorbance values as shown by the red arrows in the figure below it often indicates an improper blank was measured The green arrows in the same figure illustrate the difference in measurements after a new blank was measured 7 15 Section 7 Tools amp Configuration When this occurs reset the calibration check and ensure all pedestals are clean before making a new blank measurement Multiple Measurements with Same Aliquot Measuring the same aliquots for subsequent measurements often results in most or all of the 1 0 mm data points moving off the reported scale In the image below note that the 1 0 mm data is off the scale while the 0 2 mm data for replicate 2 is within the accepted deviation Spectra View Dats Rephcote When this occurs reset the calibration check and ensure fresh aliquots are used for each replicate Using Single Channel Pipettor Loading samples with a single channel pipettor often results in most or all of the 1 0 mm measurements being reported as more concentrated than would be expected 7 16 Section 7 Tools amp Configuration When this occurs reset the calibration check Use an 8 channel pipettor with fresh tips for each measurement Multipl
18. 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 acquisition 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 3 g l New Units gt gt eM t mM ug ml uM A q ug l Scaling from g L 0 00E 0 nM mg l Delete Selected mg ml ng l Update Selected ng ul x Units with a diamond symbol are protected amp cannot be deleted Select and drag any unit to 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 Section 3 General Operation 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 weigh
19. Viewer to close when closing out of an acquisition module User Preferences File Help Default Microarray Proteins amp Labels Protein A280 General Settings Reports Data Exporting Nucleic Acids UV Vis Sample ID required Automatic Column Advance Prompt User to Close Data Viewer Save amp Exit Cancel 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 7 22 Section 7 Tools amp Configuration 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 A280 General Settings Reports Data Exporting NucleicAcids UV Vis To export NanoDrop 8000 data automatically after each measurement check the Automatic Data Ex
20. 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 at on our website 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 i
21. 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 configurations for each method type and save multiple customized formats Eile Configuration Deta Reports Heip 432007 3 05 PM Pits Report Repon Name d ngul 600 rg 600 agul 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 Help Measure Blank Re blank Show Report 7 7 Section 7 Tools amp Configuration 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
22. brand or style with a more rigid tip structure 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 3 1 Section 3 General Operation 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
23. 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 53 Section 5 Standard Methods 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 no biologically active material is present on the measurement pedestals The metal fiber optic fittings are made from 303 stainl
24. is plugged in but not in use the power consumption is 3 W and the flashlamp is not energized Also the instrument does not utilize 2 3 Section 2 Initial Set up 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 Registering Your Instrument Please register your product We periodically update our software and add new features free of charge We would like to keep our user list updated so that we may alert you to these updates All information supplied 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 4 Section 3 General Operation 3 General Operation Loading samples on position A when operating within the Single Sample Mode Loading samples when operating within the 8 Sample Mode 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
25. 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 pedestals There are a few cases i e dried proteins that may require a more rigorous cleaning protocol For these cases we recommend that 0 5M HCI with 5 ul of dH20 to remove any residual HCI 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 9 1 Section 9 Maintenance and Warranty no more black residue To check
26. populate the report after exiting File Configuration Deto Reports Help Plots Report Fest type Report Name Repor Full Mode Piate Well Semple Oate Time na ul A260 A280 260 2 10 All 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 Eile Configuration Data Help Configure Report Plots Report Sort Report Testtype Report Name Save Report Format Report Full Mode Ignore Load Report Format i Plate Well Sa g ul A260 A280 260 280 260 230 Constant Cursor ID Save Report Pos Load Report Print Report TTT Choosing the Configure Report option brings up the following box 7 8 Section 7 Tools amp Configuration Report configuration editor Select the report columns to display and the order thatthe colurnns should appear Change the order by selecting and dragging a column header string to the desired position Report columns CE gt Well Sample ID Date Time T Move Up A280 260 280 260 230 Move Down Constant Cursor Pos Cursor abs 340 raw Measurement Type Selecting OK will return the user to the Report page dis
27. 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 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 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 Choose Standards Source Standards are required Choose the Standard Curve source Load Standard New Standards Curve Measurements Cancel 5 29 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 Meosurements Table Double Click on any row to change the concentration or delete replicates Do you wantto load the concentrations and curve type for generating a standard curve froma previously saved standard curve file Clicki
28. range of detection is 100 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 B
29. samples measured by a standard cuvette 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 Section 1 Overview 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
30. 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 5 39 Section 5 Standard Methods Measurement Concentration Range Using the regular Bradford assay the concentration
31. 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 list file before measuring samples Confirm that reference blank solution and solvent are the same material Buffers often absorb in the UV range and therefore it is critical to blank the instrument with exactly the same material that the sample is suspended in Confirm that your sample is not too dilute 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 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 8 12 Section 8 Troubleshooting 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 F Small changes in solution pH will cause the 2
32. 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 7 13 Section 7 Tools amp Configuration Note The black appearance of the removed residue is normal and is not indicative of surface dirt 5 Ensure the measurement pedestals are clean and that a 1 5 ul water sample beads up on each of the lower pedesials Repeat reconditioning procedure if a water sample droplet flattens out and covers the surface of any pedestal NOote CF 1 is supplied in two single use vials and both ampoules must be used within 30 minutes of opening Prolonged exposure to the environment may cause a significant concentration change Calibration Check Procedure 1 Position the instrument at an angle that will allow for optimal use of the tip guide 2 Select the Calibration Check Tools amp Configuration Tab gt Utilities amp Diagnostics gt Calibration Check 3 Enter the Target Absorbance of the CF 1 in the pop up box Target absorbance 350 nm 1 mm pathlength Note The target absorbance is located on the ampoule label and is lot specific 4 Add 60 ul of dH20 to each well of one of the 8 well strips provided 5 Use an 8 channel pipettor to simultaneously pipette 1 5uL of water to each pedestal position lower the arm and click the Blank button At the end of the measurement cycle use a lab w
33. without the respective measured values See image on the right above This option is very useful when running a routine series of standards 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 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 3 13 Section 3 General Operation 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 14 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 X Load Sample ID Manual Sample ID File Entry Cancel ik Load Sample ID File The NanoDrop 8000 software enabl
34. 0463 Asm 00 Semple ID e ana 4 Cl Sareat 1 pte 04 asm oo Semple ID ee Active 8 1 DI Serle 1 mias 0460 AS 00 Sanok ID Ace si N El Sengeti 1 rntaba 009 Aen U0 Semple ID am 1 a By Peay aac _________ ae 00000000000 Awel ja FI Somget 1 ran Tabs 0470 ASO 0 004 8600000000000 smmm cle0o0000000000 D Awe Si 1 GI Samplet 1 rm tabs 0467 Atm 0079 E Sampin ID __ E A m 5 asinek of Active 8 1 HI Saree 1 ren tabs 0471 ASD 0 005 c 00000000000 eo iE i HI OOOOOOCO00000 m Te 200 CPE 000a 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
35. 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 0 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 ND 8000 Data folder Note For laboratories requiring that every user have a unique user account the administrator may disable the default user account Account Log in Log out and Time Out 7 19 Section 7 Tools amp Con
36. 1024x768 required Check the computer settings Be sure that the Start menu tool bar is set to the bottom and not along the side Low Detector Bias This occurs when the software has 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 8 7 Section 8 Troubleshooting 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 Tf 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 o
37. 3 General Operation Nucleic Acid Eile Edit Configuration Help Measure Re blank C Recording ShowReporn User Default Date Time 9 9 2008 2 46 PM Cea Plate ID Make new BLANK Measurement rs E Sampie Typ i 2 All Active On Off nm1 260 Unite not 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 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 soluti
38. 60 280 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 ionic 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 Chomezynski 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 Guan
39. 659 461 0 4598 461 2 4611 4616 4569 4588 4565 4577 4569 4578 4589 4583 458 2 MEBI 4616 457 7 459 0 4592 4587 4586 4590 460 1 4602 4595 461 1 461 3 4598 460 0 457 0 4595 4571 4585 4583 4586 4642 4593 4605 4632 4597 4596 4575 4585 4565 RM 4568 4560 4593 4575 4583 4586 4595 4596 4614 4622 4603 4616 4598 4586 4648 4616 4623 4613 461 7 4608 H 4586 4599 4569 4586 4574 4590 4608 4587 4585 4605 4606 460 2 Cell Color Code Click on any cell to select de select that cell for repeat measurements White inside limits Red outside limits Light green inside limits amp selected Repeat Selected J _Close Window Dark green outside limits amp selected 4 8 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 X File Help User Default v Standard Methods User Methods Tools amp Configuration Single Sample 8 Sample Proteins amp Nucleic Acid bik Protein Protein A280 BCA Protein MicroArray aedo UV Vis Protein Lowry Cell Protein Cultures Pierce 660 nm The operating software has been tailored to meet the life scientist s needs It includes
40. A FB DNA F8 DNA F9 DNA F10 DNA FIT DNA F12 DNA G1 DNA G2 DNA G3 DNA G4 DNA G5 DNA GB DNA G8 DNA G9 DNA G10 DNA G11 DNA G12 DNA HI DNA H2 DNA H3 DNA H4 DNA H5 DNA HE DNA H8 DNA H9 DNA H10 DNA H11 DNA H12 You may choose to skip sample columns in this Plate Set by choosing a Start Column other than 1 default Start Column 1 Define Sample ID File Format Continue Cancel 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 Section 4 Sample ID Entry Options T Enter sample IDs Enter the Sample IDs via the keyboard or barcode scanner The well position increments automatically with each entry Click on a well to go to that well samples DD To de select a wall setthe replicates 10 0 D O O DODDDDDOD DODDDDDODODDDD 0o DODODDODDODDD e DODDDDDDDDDD F DODDDPDDDDDD c DODODODDODDD Next Welt Fished Canai DOPOPPPOOLHG Position Al Replicates Sample ID To move to the next well in a column enter the sample name and then
41. A sample AN ats F T DNA sample A 6 00 Absorbance 10 mm 2 00 1 00 0 00 11397 T n i n n i i T p i 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm 21 0 Chas 0 AV567 Load Next Sample ID F7 This button enables the user to automatically load the 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 10 Section 3 General Operation Functions Specific to the 8 Sample Mode Eio Edi Configuration Heip Measure Blank Rebienk Recording ShowRepot User Default Date Time 9 9 2000 221 PM Exit Plate ID 425 nul Measurement complete mii Sample Type MORA
42. BSA All Active On Oit nml 200 Units mym Ace J ni 1 A Al Sapen 7 Jabs 1 190 280 074 marmi Sampie ID BSA kii L A290 1 1 1 793 awe al A BI Samples 1 rm 1abs 1206 280280 075 maji Sample ID DSa Sek A200 1 214 1821 Ae ni n A 4 CI Saget 1 rm Tabs 1 181 ave 073 mg ml Sample ID BSA CE AZ 1 186 1 779 ace sl 1 A D1 Sapen 1 pm abs 1258 v ara gimt Semoleid BSA Net ama 9 284 1 096 340 nm Nornalizabon E Aore thoy A El S ge 1 Tabs 1 167 V 074 mg ml Sample ID 8S4 a azo 1 1 762 1 a A 12 A ase s 1 Aje Semple 1 iate 1 238 asvan 07 aint BI QOOOOOO OOOO ODO smo BSA Seti A280 1 243 1 064 CI QOOOOOOO0000 1 a Active ali A G Sample tt i nmi ade 1 106 260 2001 0 75 mg ml Eje 000090000000 0j Sem oh Ah A200 1 1 1789 FIe00000000000 ae s 1 A H sapon 1 fm Tabs 1282 26230 075 c 6 00000000000 FX majml H Samoe ID BSA ee ee M 120 1 936 20 0 CU200092 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
43. Bio Edi Conhgurahon Help Measure Blank _ Reblank Roconing ShowRepor inal Default Date Time 9ya 2000 2 20FM ea Plate ID Dichomate Automatic Path Selecton F On Mensurernent complete All Active On Ott nmi 350 5 nm2 700 gt Ade n 1 wT AT Sspet 3 mlas 0746 nmZabs 0 002 Sample ID unknown 1 A Ace 1 AF BI Sepeni 1 pm labs 0745 nm2abs 006 Sample ID unknown 2 1 ij Adme C a n NA Cl Semple ty rm 1 abs O73 ten 2abe 0 001 Sample ID unknown 3 Ko Ace C 8l 1 NA DI Sapen 1 miadb 0741 nm2aba 00M Sampel unknown 4 Normalize r Awe tt 1 M ET Spe 1 rm las O76 mmZabe 0 002 VA ajl 5 i Sample ID unknown 5 A ace t 1 MA FI Sempen 1 rm labs 0733 nm abe 0 000 BI POCOCOOO CO OOO sson wirun Sea CI QOOOOOOO0000 a petve aa AR G1 sapon 1 l misel 0735 nm2abe 0001 EIBOOOOOO OOOO O Samed _ unknown 7 j FIBOOOOOO0O00O0O poe i 1 oS a eens Seo s1 OOOS0000000 n ar 200 CPE1 008787 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 b
44. Concentration Range e eeeee eee testes teeeeeeeees 5 3 Oligo Calculators 2225 2 esint ts sheatt eee dese cette tsa gS bsitsiatfdeet sade 5 7 Protein A280 3 0 00 tinier ch os an eae Abeta ee 5 8 Sample Volume RequireMent cccceeceeeceeeeeeeeeeeeeneeeeneeseneeeeeeeeaees 5 8 Measurement Concentration Range eccecesceeeeeeeeereeeeeeseneeseaeeeeaees 5 9 MiGO AAI s 22 ix sscezhs pais canes a2 ccseadagesait te aaa aeaea EERE E a beet 5 13 Fluorescent Dye Selection ecceeeeeeseeeeeeeeeeeeeeeeesseeeeeeeeesaeeeeaeeeaes 5 13 Measurement Concentration Range ccccesceeeeceeeneeeeeeeteneeeeneeeeaes 5 14 Oligo Calculator ed heehee iene eee ete 5 17 WN VS ne cate ree ohee Bee ioe heats Oe ie tl ahaa teh A Atala A eB eset at 5 19 Sample Volume RequireMent cccccecceeeseeeeeeeeeeeeseneeeeeeseneeeeeenes 5 19 Measurement Concentration Range ccccecsceeeeceeeeeeeeeeeeneeeeneeeeaes 5 19 Proteins Label sists ssbctnd E E E AE Sotvnlda Mc A A 5 22 Fluorescent Dye Selection ecceeeeesseeeeeeeeeseeeeeeeeseaeeeseeeeaeeeseeeaes Measurement Concentration Range Protein BGAic3 sei heten Siete ei Ge ee ee Measurement Concentration Range BCA Assay Sample Preparation ccceeeceeesceeeeeeeeneeeeeeeeeneeeeeeeeeaes Protein LOWY ws e20 e ested ees tered ie S k NEN atte eee a teneee cee tes Measurement Concentration Range Modified Lowry Assay Sample Preparation cccccesseee
45. Inactive H Standard 7 oo Standard Curve Absorbance Spectra mromtmmoowory os ees oa aa eee 0 0 i i pna i T i 550 560 580 600 620 640 660 8 740 750 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 5 50 Section 5 Standard Methods 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 Standard Double Click on any row to change the concentration or delete replicates ug ml Ave Abs Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 Measure A Peference 0 000 0 000 0 001 0 001 0 001 0 001 0 000 B Standard 1 125 0 0 016 0 019 0 017 0 016 0 014 0 013 Blank C Standard 2 250 0 0 046 0 046 0
46. Member State and this product should be disposed of or recycled through 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 Section 2 Initial Set up 2 Initial Set Up Computer Requirements The operating software will only run on an IBM compatible PC meeting the below criteria No Mac versions of the software are currently available e Microsoft Windows 2000 XP or Vista 32 bit and Windows 7 32 and 64 bit e Windows Vista has also been tested successfully with the software e 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 e 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 U
47. ND8File 3 23 2007 8 57 AM en Custom report Formats 5 Default Cell Culture omm amp E Nucleic Acid User Defined Data File Export Location In addition to the primary data storage users may elect to export their data as anto 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 Data Export feature by enabling the On box Select 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 7 2 Section 7 Tools amp Configuration 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 View
48. 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
49. Position Illuminator The entire measurement cycle takes approximately 20 seconds less time if fewer than 8 positions are used Re blank F3 The Re blanking 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 7 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
50. Position Illuminator will stop flashing Unique Screen Features Eile Edit Configuration Standards Help Measure Blank _ Rebionk Recording ShowRepon Usor Date Time 9 10 2000 2 19 PM En Plate ID Lowry pasuorment complete s Standards Al Active Onyo nmi 650 Virw Updato Standards a Active E si 2 mias 01 Semple ID o _ am 0785 Active m D2 Samen 2 ran tabe S moelD Lo Asso 01 Active E C2 Saget 1 home Semple ID e i Aen 027 Actve E D2 Sanget 1 Sample ID ar i 7 AED 0214 Active E E2 Samen 1 Sample ID ee a Active E F2 Samen 1 Semple ID Lowey 5 ji ASS 0216 Active m 1 G2 Senet 1 A406 0110 000000000O sao _ tasr aeo azr 0000000000 ame 0000000000 000000000g H2 Sagen 1 A405 0142 oe Lowey Pra asso 0 217 20 0 00150 0 37 0 e A650nm the Cu complex s absorbance at 650 nm for the 1 mm pathlength e nm 1and nm 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 5 34 Section 5 Standard Methods 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
51. 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 configuration 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 10 Section 7 Tools amp Configuration 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 p
52. S All Active OOt nmi 260 Unita ngul M Actve E ni 1 bf AJ Semple 1 mm abs 8811 A20 8811 ng ul Sample ID M440 ng l Aveo ams 2607280 188 v 219 fee Aefve m t 1 AN B3 Sampon 1 rm1absl 8656 aamol aese naul SampleID 7440 ngiu Aal 475 mwl 1 86 awzal 13 ee Adve 0 ni t LAP C3 Semple tl 1 rm abs 8764 Age 6754 na ul Sample ID 440 nga A azo 472 2807280 185 20230 217 zana Active m l 1 kance Sapen 1 pmtabs 878 azo em nahl SemoleiD angia N az 4726 awal 1 06 awl 219 am Acie m i i L NE Sanok a 1 rm abs 87e A20 8782 aahi Sample ID 7440 ngra Azo 4702 28020 187 207230 213 eee Active ou 1 VAN F3 Semple 1 rmiabs 8789 azal 76 oy Sample 1D 480 ngha aol are 20 200 1 66 awl 219 ve Active m 1 LA G3 Sample i rmi abs 8 004 A200 0 004 Raka Sample ID 7440 ng A200 4710 w020 1 87 vzw 2 19 TAEA Adem 4 1 Wi H3 Sample 1 mial 8a Azo aso oy SamoklD 4e0ngia A ane 2020 107 a2w_ 213 moo 200 00580 3778 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 de
53. T 4 lf 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 6 Reconnect the power supply wait 5 seconds and then 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 Welcome to the Found New Hardware Wizard Tha wicard helps you nitah sotare for NareDiep USB Device Can Werd correct to Wraae Usdte to we arch her solte 2 11 724 hendrere come nith an inslallation CO P o Noppy Pa me oriy What do you mare Pa wnand 10 9 D Jowtad the morare aly Recomearsad Instal hors a tet or specific location ladwancect Click Newt to contirese Click Neat to contin pet Coce lt Back M yes Carcel Intro Page Windows XP SP2 Other Windows Operating Systems Note If the Found New Hardware Wizard appears installation will require two cycles through the Wizard once for the spectrometer 8 2 Section 8 Troubleshooting and once for the peripheral control devices that are internal to
54. 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 dH 0 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 8 9 Section 8 Troubleshooting Saturated Detector The detector is saturated for the following channels B This is most likely caused by
55. a spectrometer utilizing a linear CCD array is used to analyze 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 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 Section 1 Overview 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 s We have contracted with one or more recycling disposal companies in each EU
56. aaes 7 12 Calibration Check si oiean nena end Riedie ed ented eee 7 12 Calibration Check Best Practices ccceeseceeeeeeeeteneeseneeeeneeeeeete 7 12 Common Technique ISSUCS esceeeceeeeeeeeneeeeeeeeneeseaeeeeeeeseeeeaeete 7 15 Intensity Check ss csi decane ieeren ae ene E eE cies 7 18 Account Management ccesesceceeeseeeeeeseneeneseecenenseeeeseseneeseneneeneeteneres 7 18 User Preferent ESen snoer ao en ee aidan a ira aiid 7 22 B TFOUDICSHOOUING 22 cncccsece et ceseenezedieccetetececueastedesteuececsererstececedaedenees Error GOd S s ise cttain dete e aa a a Bsn iia heen ela eatin ee Instrument Not FOUNC cic Merce tebe event anette Connection Erots 2 eis RGA eee en eee SIGN Al SeA EA E sek eel rice T ET Error COd 8 eic ccestes cece tesviev eins sieves Rave E STENE E cee AE e eeii Error G0d6 80133 tac asc cee tint ied atten avis ae aa See Liquid Column Breakage Unusual Spectra Aintsescesiaceatints coh T aE Sample Accuracy and Reproducibility eceeeeeeeeeeeeeeeeeeeeeeneeeeeere 8 11 260 280 RaO n sa t aa aaaea aa er eea ra T eo anaa a a A eaaa 8 13 Technical SUppOrt 2c 2h ei en SA ae et 8 14 9 Maintenance and Warranly cccsccsseesseeeeeeeeeseeesesseeeneneeeeenees 9 1 GIOIA sa Sete iden seg E A ETT 9 1 Decontamination of Measurement Pedestals cceseeseeeeteeeeees 9 2 Rapid Reconditioning of the Sample Retention System 9 2 Calibration cc c ccd aa raaa nee ea e da
57. abs 0351 _ Dyolpmolhd 2337 ngful Sanple ID CMCS iq AA rm iabs ONT DyeZabe U6 Dye2pmold 25 50 34 88 Ade m ont tC Semple 1 Dyalate 022 Dyal pond 23 47 Dye1 o3 x ng ul K z Sample ID C35 NT rm tabs 0122 Dye aba OSIB Dye 2pmolkd 25 94 33 89 Dye2 Os Z Acim B H t t Sape 1 Dyetabs 0252 Dyelpmoll 7345 anju Semple ID O25 AL nmiohe 0124 Dye2abs 0630 Dye 2pmolul 2554 33 76 Active m t Le Semple 1 Dyelabs O52 Dye l pmol Z349 ngiul 4 E 2 12 Sample ID CyS en ae rent abe ID Dyn 2abe OBO Dyn 2pmathd 2559 33 84 A t tT Sapen 1 Dye tabs 0354 Dyn tpmolid 2357 oghul BI BOOOOOO000O0O s CO5 AA rm obs 0131 Dye Dabs 06H Dye 2pmolAk 2557 33 99 Cl BOOOO0OO00000 T 3 D fe al f Sample ti J Dyetobs 0I Dye lpn 239 Sara Ele 00000000000 Ve NA miadb ONG DyeZabe 0637 Dye 2 pmol 2550 3354 F SBOOO00O00000 ml 1 T Sample 1 Dyotabs 0252 Dyolpmat 2267 6GIBB OOOO0O0000D ng ul H Cy 6 N AAN mial 0123 Dye2ebs 0633 Dye pmol 2534 33 72 20 0 CO1SE 0 46787 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 m
58. after the module initialization is complete 5 35 Section 5 Standard Methods Choose Standards Source Standards are required Choose the Standard Curve source Load Standard New Standards Curve Measurements Cancel 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 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 JEIEWPETEES Help Load from file Measure Blank View or Measure Standards i 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 st
59. amples and or files for importing The keys can also be used to deselect multiple samples 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 Selected 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 7 6 Section 7 Tools amp Configuration e Movable x
60. anan ea edi ae 9 3 Pathlength Accuracy Calibration Check cccesceseeeeeseesnreeeneetenees 9 3 AEETIS ETa TO Ia EEE EEE T TE 9 3 Warranty oin n o ieee ene 2a 9 3 10 App nditES rssi Senne an aaneen aaan Sanae 10 1 Instrument Specifications eceeeeeeeeeeeeeeeeeeeeeeneeeeeeseeeeeeeeeeeseneeeeaees 10 1 Blanking and Absorbance Calculations cccccceeseseeeeeeteeeteeeeeeeeaes 10 1 Concentration Calculation Beers Law csccceeceeeeeeeeteeeeeeeteneeeeeeees 10 2 Generali okisi teers teeters ant carta ie Nae a E aE A 10 2 Nucleic Acid Sisar a aa a eee a 10 2 Decontamination of Measurement amp Optical Surfaces cece 10 3 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
61. andard 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 5 36 Section 5 Standard Methods Edit one standard Standard Standard 5 mg ml 2 000 Average absorbance 0 151 Abs Replicates Delete PEN Selected uaz cene 0 151 0 153 Reset OK Cancel Alternatively previously saved standard curves and standard curve concentration series may be loaded using the Standards menu bar drop down options 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 mery Standard Curves file Standards Help PrimPage z000 3 Standeed 6 4000 z H Standed 6 000 Wavelength 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 5 37 S
62. assie 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 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 Cancel 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 5 42 Section 5 Standard Methods Do you wantto load the concentrations and curve type for generating a standard curve from a previously saved standard curve
63. blem contact your local distributor or Technical Support for assistance Error Code 8 8 5 Section 8 Troubleshooting Error Code 8 Error reading or writing to file This might be caused by 1 The current Windows account does not have Read and Write priveleges to the folder C ND 8000 Data and all of its subfolders Contact your PC administrator to give all users Read and Write access to these folders 2 The log files have been removed from the folder CAND 8000 Data log files Replace the log files if they have been moved Ifthe log files can not be located reinstall this software 3 The log files described above are setto read only Check the properties on each log file and ensure that the Read only box is unchecked This error code is most likely to occur if the 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 attaching the USB cable please wait at least 30 seconds for the USB devices and internal drivers to be installed a
64. 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 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 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
65. ccount Management module establishes whether individual user passwords will expire and if so after how many days 7 20 Section 7 Tools amp Configuration Maximum Password Attempts 100 Minimum Password Length 0 Password Expiration enabled Oo Default Expiration Days 360 v OK Cancel Passwords log file This file contains the User ID amp password for all accounts and is readable only by the software It can be found in the c ND 8000 Data log 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 Data log 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 21 Section 7 Tools amp Configuration 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 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
66. 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 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
67. 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 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 Help Define Sample ID File Format sure Load Sample ID File Manual Sample ID Entry Be Sample ID Required ple T Auto Advance Columns Prompt Close Data Viewer Measurement Limits Show Plate Summary 4 5 Section 4 Sample ID Entry Options 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 description of this feature and the following message will appear when the user attempts
68. column be formed so that the gap between the upper and lower measurement 3 2 Section 3 General Operation 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 wipi
69. ctrum 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 8 14 Section 8 Troubleshooting clipboard Next paste this screen capture into MS Word MS Paint this program usually comes standard with the PC and can usually be found in the Start gt Accessories menu or other graphics programs Save this as a jpg or doc file and send as email attachment to your local distributor or to Technical Support Data Archive Files If you have questions about your data please send the archive file containing the suspect data as an email attachment to your local distributor or 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 15 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
70. d 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 Assay Approx Approx Typical Reproducibility Type Lower Upper minimum 5 replicates yp Limit Limit SD mg ml CV 15 1 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 5 46 Section 5 Standard Methods 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 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
71. dampened with dH 0 A final cleaning of all 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 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
72. dvantages of the sample retention system is that samples can be recovered from the upper and lower measurement pedestals by extraction with a pipette 3 4 Section 3 General Operation 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 s Proteins amp Nucleic Acid Labels Protein Protein A280 BOA Protein _Mary gaama Protein Cell Protein Cultures Pierce 660 nm 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 3 5 Section
73. e Aliquots Using Same Pipet Tips Using the same pipet tips for subsequent measurements often results in one or more of the 1 0 mm measurements being reported as more concentrated than would be expected See image above When this occurs reset the calibration check Use an n 8 channel pipettor with fresh tips for each measurement Insufficient Volumes When just one or two samples do not show 1 0 mm data points but do show 0 2 mm path deviation data as illustrated below the full aliquot may not have been delivered to the pedestal resulting in a broken column When this occurs reset the calibration check and ensure sufficient volumes are used for each replicate 7 17 Section 7 Tools amp Configuration Reviewing Previous Calibration Check Results The calibration check module included in NanoDrop 8000 software versions 2 2 and above enables the user to review absorbance values for each replicate for all 8 positions as well as by individual positions This feature is useful when troubleshooting calibration checks that result in conditional passes and or failures The latest software version is avaialble for download at www nanodrop com Previous results may be reviewed at any time by using the Load Cal Check File feature avaialble under the File drop down menu Additional information Cleaning instructions the calibration check procedure as well as a table of calibration check tolerances may be acce
74. e 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 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 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 Dye 1 user selected dye Dye 2 user selected dye Dye 1 or 2 abs normalized 10 mm equivalent absorbance of selected Dye 5 25 Section 5 Standard Methods 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 conce
75. e required Factor Value ng ul 1 00E 0 Analysis Wavelength nm 300 Units ng ul v Mol Weight g mol 0 00E 0 l OK Cancel 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 editing 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 6 Section 6 User Methods User Method Acquisition Screen Example custom Eile Eda Configuration Help Meesure lank Rebionk Recording ShowRepot User Detauit Dato fime anzeons 348AM Em Plote ID Make new BLANK Measurement halal Aulomane Path Selection E All Active On Of nmi 260 gt Units nyd Active a 4 tf At 2502 o Sarget 1 rm Tabs 1118 280 280 1 85 ng ul Sampi Semele ADO 1 110 A280 0 602 EKAA os m ts Analysis Wavolongth 260 A al 4 N BI Semele i m1 abs 113 ouvze0 1 80 ng ul Factor 60 00 Sompk ID Sample 2 AXD 1138 A200 0 633 560 2 Mol Weight g mol 0 000 Acwe al 1 ih Cl Senet 1 mial 1 158 vee 1 73 ng ul Sample ID Sanele 3 K AD 1158 AWD 0868 579 1 Actin it DI eC i Semple 1 emib 1 131 20200 1 8 ae Sample ID Sangle 4 b wer ADDL 1 131 Azan 0613 565 6 Acwe u 1 ih ET Sagat 1
76. e 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 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 5 20 Section 5 Standard Methods 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 5 21 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 Na
77. e51 Sample59 Sample67 Sample75 Sampe83 Sample 91 Sample 4 Sample 12 Sampe20 Sample 28 Sampe36 Sampe44 Sample52 Sample60 Sample68 Sample 76 Sample84 Sample 92 Sample5 Sample13 Sample 21 Sample29 Sample 3 Sample45 Sample53 Sample61 Sample69 Sample 7 Sample 5 Sample 93 Sample 6 Sample 14 Sample 22 Sample 30 Sample 38 Sample 46 Sample 54 Sample 62 Sample 70 Sample 78 Sample 86 Sample 94 Sample 7 Sample 15 Sample 23 Sample 31 Sample 39 Sample 47 Sample 55 Sample 63 Sample 71 Sample 79 Sample 87 Sample 95 Sample 6 Sample 16 Sample 24 Sample 32 Sample 40 Sample40 Sample50 Sample64 Sample 72 Sample 60 Sample8 Sample 95 Continue Cancel 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 PlateSet 1 Sample ID File CAND 8000 Date Plate files 3 plates Data by rows b Sample Well Position Assignments i 2 3 4 11 12 DNA AT DNA A2 DNA A3 DNA A4 DNA A11 DNA A12 DNA BI DNA B2 DNA B3 DNA B4 DNA B11 DNA B12 DNA CI DNA C2 DNA C3 DNA C4 DNA C11 DNA C12 DNA DI DNA D2 DNA D3 DNA D4 DNA D11 DNA D12 DNA E1 DNA E2 DNA E3 DNA E4 DNAE11 DNA E12 DNA FI DNA F2 DNA F3 DNA F4 DNA F5 DN
78. ection 5 Standard Methods 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 Dio Standards tiolp Pam Poge User tan 102003 213 FM Panum to Sample Acquistion TE Modified Lowry Standard Curve 0 2 4 0 mg ml Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module 5 38 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 Requirement The presence of
79. ection 5 Standard Methods 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 User Detouh 4242007 1017 AM Ratum to Sample Acquistion TETTEIT BCA Standard Curve r00 i 0404 J 0633 6000 0381 using a 20 1 reagent Standart Curve Absorbance Specs 104 Stenderd Curve Type Pobnonal 2nd order si Peaquored j 9950 pie Stnndants Help Punt Page User Moasiwemerts Table to sample volume 0 2 8 0 mg ml 035 40 45 so 55 eo 6S 70 75 ab Detout mym 42007 2 IGI Reten to Sample Acquestian DETTE Double Cick on any row to change the concertreton or delete replicates Abs t 0004 oo Abe 2 Abs 3 Abs 4 Abs 5 006 ja 0005 ome 00 a2 a ome oos oom 008 BCA Standard Curve ois 0218 0273 using a 1 1 reagent aa 1 mim T R 1 to sample volume Standard Curve Type Uunear Exiting the BCA Module It is recommended that you process all of the unknowns to be assayed before exiting the BCA software module 0 01 0 20 mg ml 5 32 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
80. eeetseeteeeeeaes 3 2 Sample Size Requirements cccccceceeeceeseeeeeeeeeseeeeeeeeeeeaeeeeeeeseaeeeeaeenaas 3 2 Sample HOMOGENEILY ceeeeeeeeeseeeeeeeeeeeeeeeeeeesaeeeseeeesaeetseeetaeeteaeeteas 3 3 Single vs 8 Sample Modes ceccceceeeeeeeceeeeeeeeeeeeeeeeeeeseaeeeeeeessaeeseaeereas 3 5 Functions Common to Single and 8 Sample Modes ceeseeeees 3 5 Functions Specific to the Single Sample Mode sceeceeeeeeees 3 10 Functions Specific to the 8 Sample Mode scceeceeeeeeeteeeeneeeeaes 3 11 Standard Guvesi e aaa aean aa e aa A a aaie 3 12 4 Sample ID Entry OptionS sssssssssunennnennnunnnnnnnunnnnnnnnnnnnnnnnnnnnnnnna 4 1 Sample ID entry Single Sample Mode ecceecceeeeeesseeeeeeeeteeeseeeeeses 4 1 Configuration Options for Single Sample Mode cseeeeeeeeeeenees 4 2 Sample ID entry 8 Sample Mode ecccceseeeeeeeeeeeeeeeeeeeeeeeeeeseaeesseeenaas 4 2 Configuration Options for 8 Sample Mode ecceeeceeeseeeeeteteteeeeeees 4 5 Sample Position Illuminator eee eee eeeeeeeeeeeeeeeeeeeeeeeeesaeeeeeeessaeeneeeenaas 4 7 5 Standard Methods sssccssseceeseeeseseeesseeeeseeeeeseeeesseeseseeeenseeesesees 5 1 Main Men en tisian urn OA taid ate lise E ativan 5 1 Nucleic Acids ene aat annaa Malet caeetotian a na Eie es lie TEESE ENEE SENESE 5 3 Sample Volume Requirements ssessssesessrereereerrsrerrernnrrsrnsrnernsrnrrena 5 3 Measurement
81. ength 10 1 Section 10 Appendices 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 is the analyte concentration in moles liter or molarity M Fluorescent Dyes The software uses the general form of the Beer Lambert equation to calculate fluorescent dye concentrations in the MicroArray module The table of extinction coefficients for each dye is below Dye Chromophore List Editor Dye Chromophore List i i Name 1 M cm g Mol 260 nm 03 1 50E 5 0 00E 0 0 00 i Below O5 250E 5 DOOE O 0 00 Alexa Fluor 488 495 0 00E 0 Alexa Fluor 546 1 04E 5 0 00E 0 0 i Scleded Alexa Fluor 555 1 50E 5 0 00E 0 y Edit Alexa Fluor 594 7 30E 4 0 00E 0 y Alexa Fluor 647 2 39E 5 0 00E 0 Alexa Fluor 660 1 32E 5 0 00E 0 O35 1 50E 5 0 00E 0 O65 2 50E 5 0 00E 0 Test 0 00E 0 0 00E 0 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 ma
82. equence See the Section below Unique Screen features for more details regarding the use of this feature as a stand alone Oligo Calculator 5 5 Section 5 Standard Methods 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 down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations 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 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 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
83. er module Data Viewer Features The Data Viewer is composed of two or three pages in a tabular form consisting of Plots Reports and Standard Curves where utilized The user may access any page by clicking on the tabs The software opens to the Report page whether accessed through the Main Menu or Show Report Note Recording rather than Start Report must be selected in order to access the Data Viewer via Show Report Tool Bar Features common to all three pages include 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 in a 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 7 3 Section 7 Tools amp Configuration
84. er 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 at the time of measurement Note This page is only available for software modules utilizing a Standard Curve File Configuration Data Reports Help 1D Plots Report Standards Test type Bradford 10 27 2005 1 17 Standards Sample Curve Ref Ref Std 1 Std1 Std2 Std 2 Std 3 Std 3 Std 4 ID Type conc Abs conc Abs conc Abs conc Abs conc Reference Interp 0 00 0 029 NaN NaN NaN NaN NaN NaN I NaN Standard 1 Interp 0 00 0 029 100 00 0 047 NaN NaN NaN NaN NaN Standard 2 Interp ooo 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 7 11 Section 7 Tools amp Configuration Note Save and rename files before making any changes if opened with Excel type of programs
85. es the user to load a list of predefined sample IDs or names 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 ND 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 Sampie IDs Emer the Sample IDs via the keyboard a Sample Alternatively a sample name may be entered directly into the sample ID box on the acquisition screen prior to each measurement 4 1 Section 4 Sample ID Entry Options 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 in sample IDs the entry operations may be acce
86. ess steel and are resistant to most common laboratory solvents see Solvent 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 Routine use of ethanol or isopropanol for cleaning is not recommended 5 54 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 Cy 5 This is a protected method Cy 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 6 1 Section 6 User Methods Method L
87. et 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 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 5 41 Section 5 Standard Methods 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 Coomassie G 250 and the Coomassie Plus Protein Assay kit from Pierce preliminary results show the Coom
88. etails 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 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 Performa 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 8 11 Section 8 Troubleshooting 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 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
89. f calibration Tf this 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 8 8 Section 8 Troubleshooting Pedestal Reconditioning Use the NanoDrop Pedestal Reconditioning Compound PR 1 asa 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
90. figuration 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 entries in the A
91. 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 EJEWEETEE 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 r Selected 0129 0 123 Reset OK Cancel v Alternatively previously saved standard curves and standard curve concentration series may be loaded using the Standards menu bar drop down options 5 43 Section 5 Standard Methods
92. gent 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 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 5 8 Section 5 Standard Methods Measurement Concentration Range The NanoDrop 8000 Spectrophotometer will accurately measure protein samples up to 100 mg ml BSA without dilution To do this the
93. he expanded view will not change the selected wavelength positions on the main acquisition page Standard Curves A standard curve is required every time a BCA Lowry Bradford or Pierce 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 3 12 Section 3 General Operation 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 j Measurements Cancel 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 Clicking on the Yes button will allow the user to import just the Standard series
94. ill 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 file Standards Help _ PrintPege User Detoutt 4 3 2007 2 43 PM Retum to Sample Acquisition Measurements Table Double Click on any row to change the concentration or delete replicates mami AveAbs Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 020 0045 0046 0044 0040 0046 0048 Bank 040 0099 0103 0099 0094 0100 0038 0 50 0120 0117 0 120 0123 0123 0m8 100 o210 02m o205 ozs 0213 0207 2 00 0355 0357 0354 0355 0356 0 352 400 0591 0593 0591 0593 0590 0586 8 00 0 923 0921 0927 0923 0922 0 920 Stondard Curve Absorbance Spectra cha KA bad B n os j _ _1 1 7 1 N jj ac i cho 06 eee eee 2 s lt Che 2 che 4 TS tT T hG 0 J hH SS SSS E REF A O PEE R EEE ERS PEE EE E 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 Sample concentrations are calculated by using 5 31 S
95. ilution the BCA assay concentration range of detection is 0 20 mg ml to 8 0 mg ml on the NanoDrop 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 Unique Screen Features BCA Protein Bile Edit Configuration Standards Help Measure Blank Rebienk Recording ShowRepon User Date Time 9 10 2000 11 32 AM Plate ID BCA nt complete Standarde AnAciva Onjot_ Units mg ml View Updete Standards Adve got Tr o O16 mg ml Semple ID BTA t 5 0 9893 Ac got we mg ml Semple ID BCA 3 1 291 Aci aod eC mg ml Semple ID BCA es J 1 050 Adve Ri 1 si mI abe OOR a mami Semole ID BCA 1 490 ti si El Sarget we oO iar mg ml SampleID BCA fs Aseel 0 068 1686 Active i
96. ine 1 15 Adenine 4 50 Cytosine 1 51 Uracil 4 00 Thymine 1 47 The resultant 260 280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260 280 ratios for the four nucleotides present It is important to note that the generally accepted ratios of 1 8 and 2 0 for DNA and RNA are 8 13 Section 8 Troubleshooting 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 a Leninger A L Biochemistry ond 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 The number is 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 zaie 300 sbo abo abo soho sho eho eho mo Teo sbo sbo Wamieng am e Application Module Screen Captures Screen captures of the actual spe
97. ine Correction Wavelengths Click on Edit Wavelengths to change wavelengths or define additional wavelengths Minimum Wavelength nm 220 Maximum Wavelength nm 750 Report 495 aj Wavelengths 400 750 Edit Report Wavelengths Cancel 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 3 Section 6 User Methods Edit method parameters Step 3 of 5 Baseline Baseline Correction Type Slope Alinear baseline correction from Baseline Correction Wavelegth 1 to Baseline Correction Baseline Correction Wavelength 1 nm 400 Wavelength 2 is substracted from ithe 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 260 280 Name Formula 260 230 gt gt gt
98. ing 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 8 3 Section 8 Troubleshooting Power Options Properties 2 x Power Schemes Alarms Power Meter Advanced Hibemate this computer Note that changing the settings below will wu Select the power scheme with the most appropriate settings for TS the selected scheme Save As Delete r Settings for Home Office Desk power scheme When computer is S Pugged in Running on S batteries Turm off monitor After 20 mins xj jAtter 15 mins fd Tum off hard disks Never x fAfter10 mins j System standby Never T After 30 mins z System hibemates Never a ater 45 mins z Defective USB Port on PC If your instrument operates properly most of the time but the Connection Error appears intermittently it could be caused by the USB port on the PC If this occurs install the software and operate on another PC If the error does not occur on the second PC it may be necessary to replace the USB card on the original PC Signal Error Signal Error This is most likely caused by Sample surface is dirty make sure surfaces are clean Sample arm make sure sample arm is in down position No power to instr
99. ing contact your local distributor or Technical Support for assistance 8 10 Section 8 Troubleshooting Sample Accuracy and Reproducibility If you are obtaining results that seem inaccurate or not reproducible it could be the result of sample or aliquot non homogeneity or liquid column breakage It may be helpful to try the following to ensure representative results Make sure the sample surfaces are clean before starting the software module A dirty sample pedestal on startup can cause erroneous absorbance readings even negative values and signal saturation It is always a good practice to clean the sample surfaces with de ionized water to remove any dried sample that might be present Note Do not use a squirt bottle to apply de ionized water e 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 d
100. initialization with dirty measurement pedestals Ifthe error persists clean the pedestals exit the software 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 a ee ee ee ee a 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 occurr
101. ion 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 with 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 on a 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
102. ions etc or in Columns first 8 sample names will correspond to the A1 to H1 positions the next 8 names to the A2 to H2 positions etc Each set of 96 samples are considered to be a plate Partial plates are allowable 4 3 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 C ND 8000 Date Plate files E lett Column is checked you must enter the Sample ID File column CN Sieh Pietainiae samme that holds the of Replicates If left unchecked of Replicates will be 1 for all samples in the Sample ID File ped Sempla ia Bete A Column 1 Column 2 Column 3 Well position Example Sample of replicates cs Sample T 3 Data Series Type In Columns A1 B1 Supe Sample 3 z Sample 4 Sample ID Column 2 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Help ofHeaderRows 1 Use Replicates Column x Sample Replicates Column 3 gt Sample to Well Position Assignments Sia Sampie Ds i 1 2 3 4 5 6 Zz 8 JE 10 ji 12 Sample 1 Sample 9 Sample 17 Sample 25 Sample 33 Sample 41 Sample 49 Sample 57 Sample 65 Sample 73 Sample 81 Sample 89 Sample 2 Sample 10 Sample 18 Sample 26 Sample 34 Sample 42 Sample 50 Sample 58 Sample 66 Sample 74 Sample 82 Sample 90 Sample 3 Sample11 Sample19 Sampe27 Sampe35 Sampe43 Samp
103. ipe to remove the water aliquots from the upper and lower pedestals and change tips 6 Thoroughly mix CF 1 Calibration Fluid by vigorously shaking ampoule and ensure all solution is collected in the bottom portion of the vial 7 Carefully break the neck of ampoule to open the CF 1 Calibration Fluid and aliquot 60 ul into each of well of the second 8 well strip 8 Use an 8 channel pipettor to simultaneously pipette 1 5uL of CF 1 fluid onto each pedestal lower the arm and click the Measure button 7 14 Section 7 Tools amp Configuration 9 At the end of each measurement cycle use a lab wipe to remove the CF 1 aliquots from the upper and lower pedestals 10 Change pipette tips then repeat step 8 Measure a total of 5 separate sets of replicates 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 TXT file in the ND 8000 Data Calib check folder on the local drive If the calibration check results in a failure or conditional pass please review each series of data points looking at trends associated with individual positions Choose the Take No Action option to review the data before exiting the diagnostic module Refer to the Common Technique Issues section below for troubleshooting information
104. ist Method Caffeine Quant Mode Standards Analysis nm 272 Min 220 Baseline Units Std Curve 350 mg l Linear Sort by Ext Coeff Method Name Factor Mol Weight LDH cell count Standards 490 230 750 cells ul Linear Note protected methods are indicated with a diamond and can only be modified by their creator Create Method Delete Selected Edit Selected View Selected Save Exit 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 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 ba
105. le 3 Dye tabs 4 506 Dye tsa 3004 any Sample ID BSA cy i A rm Tabs 5193 Dye 2obe NaN Dyn 2uM Nat 7 569 Benve a HI 2 F o i Sample 2 Dyn Tabs 4276 Dyal 2851 a SampleID BSA cy midl 4923 DyeZabe NaN Dye 2uM NaN 7340 Actve n 1 Cl Semple 2 Dyelats 4327 Dye lui 28 8 Dye1 o3 i f mg ml Sample iD BSA cys sA rm 1 abe SIF Dye 2abe NaN Dye 2u NaN 7 315 Dye2 None v aanne a dete 8l 1 DI Sapen 2 Dye tabe 430 Dye lub 2868 mg ml Baseline Type 750 nm x Sample lD BSA cy3 M A rmiahe 497 Dye2ebs NaN Dye2uM NaN 7 250 340 Bichromate H ars Ta eae EAIA a ee a Arive 1 El_s af 2 F 2 Oo ape Dye abs 4 26 Dye tum 20 64 main 7 SampleID BSA cy MAA ra ab 4 970 Dye abe NaN Dye 2uM NaN 7 238 Active a1 Fi s a 2 a 2 2847 A o N oe Dye1abs 4271 CAKE CA B Semple BSA e3 MA pm 1abs 4954 Dye 2abs NoN Dye2ua Nan 7 201 G T Active JE Gis a 2 e 4252 a o ample 2 Dye labs 4252 DyeluM 2035 mg m E Sangle ID BSA cy3 W A mmi 40 DyeZabe NaN Dye2uM NaN 7091 F a Ade ul 1 H1 Sapen 2 Dyn tabs 4231 Dawl 2821 gmit H Semple ID BSA cy3 M A pmiabs 4785 Dye2abi NaN Dye 2u NaN 7110 20 0 C0200 0 09 87 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 the sample typ
106. mF cc Sarge 2 mg ml SangelD BCA n A562 1641 00000000000 Oo L E GI samen 1050 aN 00000000000 oa a fen 1 60s 00000000060 ae 600000000000 3 y mg ml 600000000000 a ASSEL 0 074 1 859 200 00200003787 e A562nm the Cu BCA complex s absorbance at 562 nm for the 1 mm pathlength e nm 1and nm 1 abs current value of the user selectable wavelength cursor and corresponding absorbance value for a 1 mm 5 28 Section 5 Standard Methods 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
107. me 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 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 5 27 Section 5 Standard Methods Measurement Concentration Range When using a 20 1 reagent to sample volume d
108. meters associated with the oligo sequence When the Oligo Calc sample type option is first selected the following box will appear Oligo Property Calculator x Oligo Seq AAATTTCCCGGQ Nucleic Acid DNA v Modification Weight o0 Phosphorylated Double stranded C Mol Weight g mol 3 645E 3 GC 50 00 Ext Coeff I rmal cm 1 280E 5 Conc Factor ng ul 28 48 of bases 12 OK Cancel 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 Section 5 Standard Methods 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 5 18 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 sam
109. 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 when running a custom method User Preferences Eile Help User Default Microarray Proteins amp Labels Protein A280 General Settings Reports Data Exporting Nucleic Acids UV Vis Auto Reporting Select Custom Report Format Single Sample O 8 Sample Nucleic Acids VI Protein A280 UV Vis x Protein BCA lv Microarray V Protein Bradford M v v v v Proteins amp Labels Protein Lowry Cell Cultures Protein Pierce 660 nm Custom Report Standard Nuclei 1 report format nf8 MicroArray Standard MicroArray report format nf U Standard UV Vis report formatnf8 Protein A 280 Standard Protein A 280 report format nf Cell Culture Standard Cell Culture report format nf8 Proteins amp Labels Standard Proteins amp Labels report format nf8 BCA Protein Standard BCA Protein repo rtformatnf Bradford Standard Bradford report format nf8 Lowry Standard Lowry report format nf8 Pierce 660nm Standard Pierce 660nm report format nf8 Custom formulas nf8 Save amp Exit Section 7 Tools amp Configura
110. nd recognized Report Format Error There was an error loading the report format 8 6 Section 8 Troubleshooting This error occurs when the user does not have Write access to one of the report format files located at c NWD 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 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 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 Error 8005 This error occurs when trying to load a plate file that is not in a txt format 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 Error 9003 This error indicates that the monitor resolution is below the
111. ndard Methods 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 measured before the standard curve may be generated It is advisable to use use the dye reagent without any protein added as both the Blank and the 0 reference sample Note This is unlike the other colorimetric assays on the NanoDrop 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 Now Standards Measurements The
112. ng of the upper and lower measurement pedestal with a dry laboratory wipe is highly effective in eliminating carryover for samples differing in concentration by as much as three orders of magnitude see 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 3 3 Section 3 General Operation 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 Sample Recovery One of the a
113. ng 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 Standards Help Load from file al 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 5 30 Section 5 Standard Methods Edit one standard Standard Standard 4 mg ml 1 000 Average absorbance 0 249 Abs Replicates Delete Selected Reset Standard OK 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 w
114. ninstall all previous versions of NanoDrop 8000 software using Add Remove Programs BEFORE 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 C NANODROP 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 2 1 Section 2 Initial Set up To properly install the operating software 1 Close all programs and make sure that the USB cable is unplugged 2 Insert the operating software CD in the CD drive of the PC The software installation menu should appear automatically If 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 3 After software installation 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 4 Installation will require two cycles through the Found New Hardware Wizard once for the NanoDrop 8000 Spectrophotometer and once for two device
115. nipulated 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 10 2 Section 10 Appendices The generally accepted extinction coefficients for nucleic acids are e Double stranded DNA 50 ng cm ul e Single stranded DNA 33 ng cm ul e RNA 40 ng cm ul For the NanoDrop 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 HCl
116. noDrop 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 corrections 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 1 M cm g Mol 260 nm 03 1 50E 5 0 00E 0 0 00 i Below O5 250E 5 100E 0 0 00 Alexa Fluor 488 0 00E 0 O00 0 00 peer Alexa Fluor 546 1 04E 5 0 00E 0 glade Alexa Fluor 555 1 50E 5 0 00E 0 y Edit Alexa Fluor 594 7 30E 4 0 00E 0 Alexa Fluor 647 2 39E 5 0 00E 0 Alexa Fluor 660 1 32E 5 0 00E 0 O35 1 50E 5 0 00E 0 O55 2 50E 5 0 00E 0 Test 0 00E 0 0 00E 0 Note predefined dyes are indicated with a diamond and cannotbe modified 5 22 Section 5 Standard Methods Sample Volume Requirements Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly
117. ntration 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 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 5 26 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 Volu
118. ntrations for each standard This box is also used to delete a single absorbance replicate value or reset the entire standard Edit one standard iccosonam Tatio Trake Och on ery rss age te Conconouten of Gate rps Standard Standard 4 mg ml 750 0 Average absorbance 0 120 moot tear 4 1 4 Abs Replicates Eo Selected 0119 _ are 0 123 a Standerd y C Ceara 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 5 49 Section 5 Standard Methods Pierce 660nm Standard Curves File Standards Help Print Page Default 9 9 2008 4 03 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 concentration or delete replicates Standard Abs 1 Abs 3 Abs 4 Abs 5 Active IN _A Reference E d0 0 001 0 001 Active I B Standard1 oor 0 014 Active E C Standard 2 0 046 0 046 Blank Active E D Standard 3 0 092 0 091 Active E E Standard 4 0119 0127 Active E F Standard5 0 175 0175 Inactive G Standard 6
119. odule the last value of the constant entered within the Constant Sample Type will be retained See Concentration Calculation Beer s Law in the appendix for more details on this calculation nm 1 and nm 1 abs current values of the user selectable wavelength cursor and corresponding absorbance value fora 1mm 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 Dye 1 user selected dye Dye 2 user selected dye 5 16 Section 5 Standard Methods e Dye 1 or 2 pmol ul concentration based upon selected dye s extinction coefficient See Concentration Calculation Beers 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 para
120. on on all pedestals using the Measure button F1 The result should be 8 spectra with relatively flat baselines 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 3 6 Section 3 General Operation 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
121. playing only the columns of interest 7 9 Section 7 Tools amp Configuration 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 report 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 Full Report report into the Data Viewer at a later date Choose this option to export a tab deliminited textfile of the 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
122. ple range gt 2 4 pmol ul 2 5 Alexa Fluor 488 and 0 50 215 sample range 0 50 8 0 pmol ul 0 50 Alexa Fluor sample range gt 8 0 pmol ul 2 5 594 Alexa Fluor 0 42 145 sample range 0 42 6 0 pmol ul 0 42 546 i sample range gt 6 0 pmol ul 2 5 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be set 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 5 14 Section 5 Standard Methods 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 5 15 Section 5 Standard Methods Unique Screen Features file Edi Conhgurahon Help Measure _Biank Reblenk Roconing ShowReport User Default Date Time 9 9 2000 315PM Ext Plate ID Measurement complete Sample Type ssDN4 33 All Active On Ott nmi 260 Unts nguli Awe m n 1 ak Sompke m1 Dye Tabs O51 Dye l pmol Z340 ngful Sample ID G35 AA mmia O11 Dye2abs 0638 DyoZpmolhi 2953 33 19 Aco m S t 7 Sapeti 1 Dye l
123. ples 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 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 5 19 Section 5 Standard Methods Unique Screen Features
124. port 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 button Automatic Data Export x Export Data By Row Test Module Data Export Folder C Documents and Settings dave lach MicroArray Desktop UV Vis Protein A 280 Cell Culture Proteins amp Labels BCA Protein aang i Bradford onfigure Data Le Export for Module cowry Pierce 660nm Custom Save amp Exit Cancel Selecting the Configure Data Export for Module button will bring up the following pop up box 7 23 Section 7 Tools amp Configuration Nucleic Acid Data Export Configuration Select the data export fields and the order thatthe fields should appear Change the order by selecting and dragging an item to the desired position Optinally enable the export of the raw absorbance data range and log interval Export Fields Abs Spectrum Export 2 Show All Sample ID Disabled ZE Move Up Time ngul jes Move Down Amex 350 A280 260 280 Delta A 1 amp 260 230 7 Delete Constant Cursor Pos Cursor abs Amin 220 Cancel 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
125. quid columns break during measurement 9 2 Section 9 Maintenance and Warranty 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 wavelengths 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 plan 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 3 Section 10 Appendices 10 Appendices Instrument Specifications Sample Size 1 microliter Sample Number up to 8 Path Length 1 mm with auto ranging to 0 2 mm Light Source Xenon flash lamp Detector T
126. r oe A 7 Active m 8 1 F3 SS 4 7 5 z A m LA e argie 1 rm Tabs 8763 A250 8763 aM B Sample ID 440 ngid AZ 4 706 2607280 1 85 2230 213 r Active s G3 sa 2 D a MA anget 1 rn tabs 0004 A260 8 004 ng ut E Sample ID 440 rgd Az 4 710 260 280 187 2072730 219 oz F Hie Fs ENE 1 Active m 2 1 HI Sagat i rm1 abil 0 902 A20 0 002 G wre at ngful Sample ID HO rgd AO ATIG 250280 187 v 213 n e e 000000000 me 20 000150 03740 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 s
127. radford Assay 5 40 Section 5 Standard Methods Unique Screen Features Ble Edt Conhqurahon Standards Help Measure Blank Reblenk _Roconing _ShowRepon User Default Date Time 9 10 2000 11 01AM Ext Plate ID Bradtord Measurement complete Senders O OOOO All Active On Oit nmi 595 Units ugi Lew Jpdato Standards avasid Ade nii Al Semple rm 1 abs G08 wre W027 can angle 4 asoa 560 1 deme 8 1 BI Samples 1 rm labs 0076 A750 0 023 daliai Sample ID H A595 00 457 0 1 1 amp 7 k T Acie CI Semple 3 mial 007S aral om ugm Sample ID H ASS GUIS 486 2 dete 8i 1 DI Sample 1 rmi 0079 A750 1008 ug ml smole ID an Ass 0079 403 3 Aive a El Samples 1 rm1 abs 082 Ara O07 dnl Sample ID m m ASS 002 527 8 1 a By 12 j A dete J u 1 Fl Sample 1 mias 0079 amoj 0008 wot BIPQOOOOOO OOOO OD smon _ A535 0079 400 6 cje 00000000000 n a 5 D Active C 1 Sample ti fm 1 abs 0 003 ATOL 026 ugm EI QOOOOOOO0OOODO 0 FAS _ 0 009 bana F SAQOQOQOOQTQTOQ Dli il n AEE i EA en i c j 6e00000000000 nara Hl Semole ID preme A5SS 0078 467 6 20 0 CPET 0077327 e A595 nm protein dye complex s absorbance at 595 nm at the 1mm pathlength e nm1andnm1 abs current value of the user selectable wavelength cursor and corresponding absorbance value for a 1 mm pathlength The wavelength can be s
128. 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 5 3 Section 5 Standard Methods has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing Section 5 Standard Methods Unique Screen Features File Edit Configuration Help f I Measure Blonk _ Reblank Recording ShowReport User Detoult Dote Time 9 9 2003 221 PM Exa Flate 1D 425 ngu Measurement complete m Sample Type M AJ Active OvOtt nmi 260 Units ngid Active m 5 1 Ai s 4 Jobs 8811 260 881 i AN anple pm 1 aba A n i SampleID 440 rg d ii ADOL 4743 260 280 1 86 avzal 219 i as kaen Active m 5 1 Ab B3 5 2 2 ky Sample t 1 pmiabe 8858 AZ 8858 ng ul saple lD HO nah AZO 4 765 260 280 1 86 200 230 219 ee a chon m 2 4 i Fak CI Saget i emia 8764 Aveo 8754 MaNi Semple ID HO rng S AD 4725 2607200 1 05 BAW 217 Marvat as ee ce ai J Active m 2 1 Pa Se DI Sarget q rm Tabs B78 Agel 8781 Semple ID HOrgi A Azan 4726 2607200 1 96 20 29213 z T a Adwa m amp 1 f E i ra 782 ave LA ence 1 rm Tabs 8 782 ADA 8782 natal Semple ID 440 rgd AD 470 260 200 1 97 22W 219 i E ry emes t Ia eo te
129. 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 5 33 Section 5 Standard Methods 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 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
130. rml 1 069 ZNW 1 76 ag ut SompleID SercleS A260 1 068 A20 0 608 534 3 1 ad a 12 Aotve a4 i Fl ig O A Sanget 1 pm taba 1155 252m0 183 Bani SampleID SenceS JU nn Al iss ADOI 0 631 5774 Adere a i GI s m T abe 2 o iA arget 1 miaa 109 INO 182 ngi Semple ID Sargia 7 A 1119 Az 0614 559 6 Aden ata jf HI 5 7 7 O arcet T rm Tabs 1073 avzal 1 77 Eni Sample ID Sargia 8 AD 1073 A200 0 609 536 7 The analysis wavelength and associated factors 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 7 Section 7 Tools amp Configuration 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 applicat
131. s gt Appearance Additional step for Windows XP click on the Advanced button 2 From item list select icon 3 Select the MS Sans Serif western font and select 8 point size 4 Click OK Choosing an alternative font may result in some text being truncated in the operating software window Software Upgrades Periodic upgrades are made to the operating software and are available for download See our website for the latest available software version 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
132. s are indicated with a diamond and cannot be modified 5 13 Section 5 Standard Methods Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous nucleic acid samples containing incorporated fluorescent dyes However if you are unsure about the surface tension properties of your sample or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample 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 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 Detecti Approx Sample on Limit Upper Typical Reproducibility Type pmol ul Limit minimum 48 replicates pmol ul SD pmol ul CV Cy3 Cy3 5 Asa ys jai m sample range 0 25 4 pmol ul 0 25 Alexa Fluor sample range gt 4 0 pmol ul 2 5 660 ey tena ae sample range 0 17 2 4 pmol ul 0 17 Fluor 647 i sam
133. s internal to the instrument 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 i Computer Management Local S USWLM DALASH BL System Tools Nl Batteries Event Viewer by Biometric ga Shared Folders 1 Computer Local Users and Groups Se Disk drives i Performance Logs and Alerts Display adapters Device Manager DVD CD ROM drives Sy Storage ig Human Interface Devices Removable Storage Gy IDE ATA ATAPI controllers Disk Defragmenter amp IEEE 1394 Bus host controllers Disk Management gt Keyboards EJ Services and Applications Mice and other pointing devices Be Modems Monitors NanoDrop Devices ND 8000 Peripheral Control Device ND 8000 Spectrophotometer B Network adapters LED Position Indicator 4 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 2 2 Section 2 Initial Set up 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 Propertie
134. s listed below Lower Approx Typical Reproducibility Sample A Tvpe Detection Upper minimum 5 replicates yp Limit Limit SD mg ml CV sample range 0 15 5 mg ml Purified 0 15 mg ml BSA paang SU mgm sample range gt 10mg ml 2 5 sample range 0 25 4 pmol ul 0 25 Cy3 Dey 100 uM sample range gt 4 0 pmol ul 2 5 5 23 Section 5 Standard Methods 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 5 24 Section 5 Standard Methods Unique Screen Features Eile Edi Conhgurahon Help Measure _Biank_ Reblonk _Rocoming _ShowRepon User Default Date Time 9 9 2000 327PM __Ex Plate ID 10 piatos DNA Measurement complete p e Sample Type BSA All Active On Oit nmi 200 Units mym Ade ni 1 Al Semp
135. s 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 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 25 dsDNA sample range 2 5 100 ng ul 2 5 ng ul 3000 RNA sample range gt 100 ng ul 2 5 2400 ssDNA 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
136. sed upon the Quantification Mode selected Note Methods requiring standard curves are not available when operating under 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 v a Section 6 User Methods Edit method parameters Step 1 of 5 Measurement Type woe The concentration is calculated Quantification Mode c A oxExt Coeff v by dividing the absorbance A rs J atthe Analysis wavelength by Chromophore Alexa Fluor 488 v Extinction Coefficient times the path length Both the Analysis _ nm and the Extinction Coefficient Extinction coeff molcm 7 100E 4 are required parameters The concentration will be returned in the selected Units For weight based units a Molecular Weight is required Analysis Wavelength nm 495 Units mM z 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 acquisition and automatic inclusion in reports The Wavelengths list is pre populated with the Analysis and Basel
137. seeeeeeeeeaes 5 35 Protein Bradtord EESE A A A TE 5 39 Measurement Concentration Range cccesceeseeeeseeeeeeeeeteeeeneeeeaes 5 40 Bradford Assay Sample Preparation c ccsceeeeceeeseeeeeeeeeneeeeeeeeeaes 5 41 Protein Pierce 660 NM ar aeeie hla eee ace 5 46 Measurement Concentration Range cccccceeeceeeseeeeeeeeeseeeeneeeeaes 5 46 Making Pierce 660 nm Protein Measurements 5 48 Gell Cultures ivi c0 0e ot sleet di ieee 5 52 Cell Suspension Concentrations ccecceeeeeeeeneeeeeeeteneeeeeeteeeeeeetes 5 54 Sample Homogeneity ccccceeseeceseeeeneeceaeeseeeseaeeseaeeeeaeeseseeeeeeees 5 54 6 User Method Snia esac ccc ieaie o eee e aiaiai aeaa aae eis 6 1 Method Editor eiii ae aaien aaae Eee a aa ES 6 1 Create New Method ec cave niente aie eet te 6 2 Edit Selected Method v 04 Gv iidr indise ein ae 6 5 View Sel cted lissi ei reoaine aiaa aaaea aE aE S iid ehh 6 6 User Method Acquisition Screen cecceeeseeeeseeeeeeeeneeeeeeseeeseeeeeaees 6 7 7 TOOIS amp Configuration cctecceeeeeeeeeee sete eeseeeeeeeeesenesesneenenseeeenees 7 1 Archived Daties eaaa ea a aae ao iaaa iiaae a DETARE a Eaei padae 7 1 DEIRA E E A E Ieee da 7 2 Import Page o eia r aaaea taa aa tana aaa aaa a tien Ala el ea 7 4 Plots Pagerie a aaa the a a aa 7 6 Report Page a e a ar re aaa PEE Ee AREORA EARSTE 7 7 Standards Paga cacao enn 7 11 Diagnostics and Utilities 0 ee ee eeseeeeesneeeeeeeeeeeeneeeeesaeeeenenaeeeee
138. sing the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations A280 10 mm Path 10 mm equivalent absorbance at 280 nm for the protein sample measured A260 280 ratio of sample absorbance at 260 nm and 280 nm 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 Show Report formatted for 200 samples although the buffer size can be modified 5 11 Section 5 Standard Methods 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 5 12 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
139. ssed from the Configuration drop down on the main acquisition page In addition the drop 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 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 4 2 Section 4 Sample ID Entry Options folder at C ND 8000 Data When creating a file
140. ssed 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 Serial Number USBZGIZ1USCPET Pedestal Dotocior Bios 364 UV Spectra amd smo amo Crops 2m0 200 Van MN SS ehegration Times c m7 atl hye n a i n ge n n n ni 20 7910 ado 2500 2600 20 200 200 00 mho Xio 300 dO MO Wavelength nm Visible Spoctra 41000 zmo sr Max Sage 26000 fa gt 2000 1mo 4 4045 nmi 2605 p25 nm 3621 O2Z32em 023l xanon Penks Montorad 10000 mo karaa i i n n n i go she zbo wbo eho sho sho Soho eho 7000 THO MMO HHO Woveleesgh nen Contguration 009 40 Account Management The Account Management module provides options for directing where specific data files are archived allowing users to segregate their data into 7 18 Section 7 Tools amp Configuration personal folders The Account Management module is accessible to the administrator only User Access Manager Actions All Users UserID Full Name Active Locked Expired Expires Default user 0 Active NotLocked NotExpired Never Administrator Administrator Active NotLocked NotExpired Never Account Types There are three types of user accounts Level
141. status color code displayed on the software screen and the pattern of illumination is determined by plate configuration at set up If measurement limits have been defined wells that are out of the defined range will flash on the 4 7 Section 4 Sample ID Entry Options 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 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 Plate ID 425 ng ul Plate Results ng ul Les p 3 4 5 6 2 e 9 at Paes 456 2 QIN 4596 4007 4603 4600 4603 4589 4580 4595 4586 458 8 4585 4609 4609 4612 4596 4606 4
142. t 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 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 9 Section 3 General Operation Functions Specific to the Single Sample Mode T Nucleic Acid Single Sample File Edit Configuration Help f e Measure _Blonk Reblenk Recording ShowRepor User Detouit Date Time 8 19 2009 402 PM Ext Measurement complete Sample D ONA sample 6 Measurement Results Units ngl S A280 0 779 nmlebs 1 448 260 280 1 86 Sop voo SON pre0 Taas 2snj30 20 ngful 724000 nmi 260 Overlay control Gear graph now 4 Legend 11 28 t DNA samples AN 10 00 DNA sample5 AN ail 9 00 i DNA sampled AY te Fes DNA sampla AN DN
143. termined by plate configuration at set up Status The Status button will turn green during a measurement cycle Indicates number of replicates Indicates actual number of Units specified in plate set up replicate measurements made j Protein A 280 File Edit Configuration Help Measure Blank _ Re blenk Recording User Default Date Time 8 15 2008 8 55 A ea Plate ID Measurement complete j Sample Type METASSENSmE v 1 280 Units mg ml i Adve 1 N Al Sample tt 2 rm tabs 1016 avas 1512 sate SampleID test A200 10 16 3 11 Section 3 General Operation 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 t
144. the instrument 7 Install the instrument on another PC to rule out a faulty USB hub port on the original PC Windows Device Manager on the 2 PC as described in step 3 above If the instrument is still not recognized then the instrument may need service Contact your local distributor or Technical Support for assistance 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 lf 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 reconnect the instrument If this occurs the USB cable will need to be disconnected reconnected before select
145. 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 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 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 li
146. the following pre configured application modules e Nucleic Acid concentration and purity of nucleic acid e Protein A280 concentration and purity of purified protein e MicroArray dye incorporation concentration and purity of nucleic acid e UV Vis general UV Vis measurements e Cell Cultures absorbance light scattering measurement of suspended microbial cells e Proteins amp Labels concentration of dye labeled proteins conjugates and metalloproteins e Protein BCA protein concentration using the BCA assay e Protein Bradford protein concentration using the Bradford assay e Protein Lowry protein concentration using the Modified Lowry assay e Protein Pierce 660 nm protein concentration using the new 660 nm assay 5 1 Section 5 Standard Methods Note Colorimetric applications are not available in the single sample mode and the associated menu buttons will be grayed out 5 2 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 propertie
147. tion 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 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 Data log 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 Data log 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 25 Section 7 Tools amp Configuration 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
148. 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 Diagnos EA Select Diagnostics or Calibration Check Calibration Check Main Menu 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 K2Cr207 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 in a failed calibration check on one or more pedestals Calibration Check Best Practices e Do not open the vial of CF 1 until ready to perform the calibration check e Position the instrument at an angle that will allow for optimal use of the pipette guide e Ensure the NanoDrop 8000 is not situated near an air vent or an exhaust fan from a nearby instrument 7 12 Section 7 Tools amp Configuration e Aliquot 60 uL of CF 1 into each of the 8 t
149. to make a sample measurement A required Sample ID field is empty 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 8 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 Measuremeni 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 6 Section 4 Sample ID Entry Options Enter Measurement Limits Enter the Minimum and Maximurn Concentration Sample measurements that fall outside this range will be highlighted in displays a Min Concentration 10 00 a Max Concentration 3000 Continue Cancel e Show Plate Summary Allows the user to review the results of the
150. ubes in the PCR strip A volume of 60 ul in each PCR tube is required to reduce the effects of evaporation and potential fluid concentration e Use asmall volume 0 5 10 uL multi channel pipettor and well fitting low retention tips If using an electronic pipettor do not draw up large volumes to dispense multiple aliquots The CF 1 reserve stored in the tips may begin to concentrate affecting the quality of results Draw and dispense individual 1 5 uL aliquots for each measurement cycle e Ensure that adequate volumes of CF 1 are being pipetted onto the center of each pedestal If tips are not fitted properly on each channel of the multi channel pipettor individual channels may not properly draw up or dispense an adequate volume onto each pedestal Columns that are too thin or shifted off to one side may not completely cover the fiber optic center of each pedestal e Always use fresh aliquots for each replicate set of measurements Required Materials e CF 8 Calibration Kit Cat CHEM CF 8 e low volume 8 channel pipettor with the appropriate tips e dH20 e low lint lab wipes Preparation Follow the cleaning and reconditioning steps prior to 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 3 Wait 30 seconds for the PR 1
151. ument check 12 power supply For more details refer to 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 8 4 Section 8 Troubleshooting shown below This indicates that the USB communication is normal the power supply is operational and the flashlamp is functioning Sorini Number USBZGIZIISCPEI Pedestal ag i A re Zo zo mbo sho ho zbo abo 200 who ndo xio whe bo mho Wevaleng s am Visible Spectr ona soene Wavelangh Accuracy Max Single Ofset 054 Monte 845 1008 3 Srm 2 Sho sbo so abo sho eo a sho esto mbo THe sO sdo 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 volt ohmmeter to the outlet of the supply The voltage should be 12 20 Vdc center positive If none of the troubleshooting steps above solves the pro
152. 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 3000 generating a standard curve from a nove gt 3000 previously saved standard curve file cave 4500 6 000 Section 5 Standard Methods 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 ina 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 generate 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 conce
153. 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 Unique Screen Features File Edt Configurotion Standards Help Il Measure Blank H 5 Recording _ShowRepon User Detault Date Time 9 9 2008 436 PM Picte ID Meosurement complete All Active On O 50 gt i j Standards ctive OnO nm Units ugm ewtuedte Standards Noli Active af 1 Sample i rm 1 abe O00 ao 0006 ways Pierce 660 A660 1 001 ooo s 1 B3 Sample 1 A750 00m gil Pieren BED HE ABD 07 45 1 C3 Songket pret Ferco kU ASU 289 1 1 D3 epia 3 ug ml j Ao 0083 E3 Songet Active af 1 Semple iD Pierce 660 Active al 1 Semple IO Pierce BAD AT ao Sample D Froe 650 20 0058 03W23 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 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 unknown 5 47 Section 5 Sta
154. 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 List Name 1 M cem Ji g Mol 260 nm 280 nm factor factor 03 1 50E 5 0 00E 0 o 00 0 00 Below 05 2 50E 5 0 00E 0 000 0 00 Alexa Fluor 488 0 00E 0 0 00 0 00 Alexa Fluor 546 1 04E 5 0 00E 0 i Selected Alexa Fluor 555 1 50E 5 0 00E 0 y d Edit Alexa Fluor 594 7 30E 4 0 00E 0 j Alexa Fluor 647 2 39E 5 0 00E 0 Alexa Fluor 660 1 32E 5 0 00E 0 O35 1 50E 5 0 00E 0 O55 2 50E 5 0 00E 0 Test 0 00E 0 0 00E 0 Note predefined dyes are indicated with a diamond and cannot be modified 7 26 Section 8 Troubleshooting 8 Troubleshooting Error Codes Instrument Not Found No ND 8000 was found This error might appear upon software startup and usually indicates that either the power supply or the USB cable is not properly connected or the software is not loaded properly To troubleshoot do the following 1 Check that the power supply is connected to the instrument
155. xactly 10 fold different as readings are dependent on both the optics of a specific spectrophotometer as well as the cell type in suspension The Cell Cultures module displays the sample spectrum from 250 nm to 700 nm 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 5 52 Section 5 Standard Methods Unique Screen Features File Edit Configuration Help Meosure Blonk Re blenk Recording ShowReport User Detoult Dote Time 9 9 2008 327 PM EA Fiate ID InrSpec Measurement complete AllActive On Off nmi 340 S Active 8 1 Al Sample tt 1 rmiaba 0 877 Aso anm Sampe ID Acie 81 BI Senge 1 rmiaba
156. ype 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 Weight 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 Included in system software compatible with Windows 2000 XP Vista 32 bit and Windows 7 82 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 Intensitybtank Thus the measured light intensity of both the sample and of the blank are required to calculate the absorbance at a given wavel

NanoDrop 8000 Spectrophotometer V2.2 User Manual

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