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NanoDrop 8000 Spectrophotometer V1.1 User`s Manual

1. ES ap Ch H OL pr EE Lesben aoe Bif A I mnm L SER ve DE Ge E Se E E Se iE SSE H I EL pp be SS Er 450 460 500 520 540 560 580 600 620 640 660 680 700 720 750 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 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 Section 11 Protein BCA 4242007 1017 AM to See cle Acquistion 10C0104 Onna eee eee ae eae v EE ded EL 2E nir 15 Tee Lee BCA Standard Curve 023 om azo o ie 0619 1 000 KA omi 1 0259 il using a 20 1 reagent to sample volume 0 2 8 0 mg ml 101 2C00140 10 208 uL 003 0 c 0019 0015 E E 38 38 38 38 E73 ETE ETE ee ert qe eem BCA Standard Curve using a 1 1 reagent to sample volume 0 01 0 20 mg ml Exiting the BCA Module It is recommended that you process all of the unknowns to be assayed before exiting the BCA software module RE 12 Protein Lowry The Modified Lowry Protein
2. 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 17 2 Power Options Properties 2 xl Power Schemes Alarms Power Meter Advanced Hibernate STE Select the power scheme with the most appropriate settings for this computer Note that changing the settings below will modify the selected scheme Save As Delete m Settings for Home Office Desk power scheme When computer is 1 Plugged in Running on batteries Turn off monitor After 20 mins DI After 15 mins Turn off hard disks Never Y After 10 mins m System standby Never DI After 30 mins System hibernates Never m After 45 mins Cancel 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 instrumen
3. deep case ett o me Intro Page Windows XP SP2 All Windows Operating Systems 4 Installation will require two cycles through the Found New Hardware Wizard once for the NanoDrop 8000 Spectrophotometer and once for two devices internal to the instrument For the NanoDrop 8000 to operate successfully a total of three USB devices need to install although only two cycles through the hardware wizard will be observed To confirm installation view the Windows Device Manager as shown below 2 1 Section 2 Initial Set up Computer Management Local ER System Tools tuj Event Viewer Ki Shared Folders Computer 87 Local Users and Groups zeg Disk drives di Performance Logs and Alerts fd Display adapters JB Device Manager a A DVD CD ROM drives yj Storage bi Human Interface Devices Removable Storage IDE ATA ATAPI controllers Disk Defragmenter t IEEE 1394 Bus host controllers Ej Disk Management X Keyboards dn Services and Applications H 3 Mice and other pointing devices Ba Modems ig Monitors k NanoDrop Devices LED Position Indicator S ND 8000 Peripheral Control Device ND 8000 Spectrophotometer HB Network adapters Your NanoDrop 8000 Spectrophotometer should now be ready for operation If the software does not start properly refer to the Troubleshooting section for possible solutions Configuring the System Font The software is designed to look best with th
4. eo ar MN l Dyna 119 009 ae 75 6 y 1 i H 7A haee d L me ngul Sage Akai CH Ai femmt TITIE 2900 we 35 3 VU DEO U XE a H eooooooooooo e Sample Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 for RNA ssDNA 33 for single stranded DNA or Other for other nucleic acids The default is SSDNA 33 If Other is selected the user can select an analysis constant between 15 150 When navigating amongst the three 3 general sample types within the MicroArray module the last value of the constant entered within the Constant Sample Type will be retained See Concentration Calculation Beer s Law in the appendix for more details on this calculation e nm 1 and nm 1 abs current values of the user selectable wavelength cursor and corresponding absorbance value for a 1 mm pathlength The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e Dye user selected dye e Dye pmol ul concentration based upon selected dye s extinction coefficient See Concentration Calculation Beer s Law in the appendix for more details on this calculation e ng ul concentration of nucleic acids in the sample calculated using the absorbance at 260 nm minus the absorbance at 340 nm i e normalized at 340 nm See Concentration Calc
5. ina Im 1 H1 Sample B li mibe o ODE AUR MaM m m Sangie fi ABES MoN MaN Standards foie E 1 Cl Gmie naiba Or AO Mei memi 3 H Hai EE cbeg Ll ma 1 Di Eege H fi es ba OO Ath Mah view Upeints Standards mum je See EI ASI MaN Mal Bebe 1 El Segen n Malam OD ATUS NoN mol Zwei EI ASRI Mai Hai 1 Fei Dd 17 tange ASE Hah Hint Gl Sample E D rel sa Of AGAS Mak mg ml Bowe LJ E 1 Fl ape H i miles OC Jug Mahi mg ml ASB Ma Han H1 Bamgie E H nx HE d Hei many Noli e A 562nm the Cu BCA complex s absorbance at 562 nm for the 1 mm pathlength e nm 1 and 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 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 e Show Report formatted for 1000 samples although the buffer size can be modified 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
6. Lamia D Den Tee l l jt irte Bi Aul s i read DA Tires ITI SH AR ik Selech rPI 81 raul DA 11 0 05 B EO Aba d 2 748 53 ui DA Ka Spe CA IL DEN AM e 12 740 ng s CINA lt lt lt Desetost Dess 5 AAD TIPO SEM ARM THEODOR B bi AR Ti amp 17B8 AEbrm ui DP Search Haid diwa Conti br Chl land de near rulela mmole 187 0 RER Search ia neiiched to Ba Ti r Se Et l i Gre ER La Imp a Retum 3 a Get E The Plots page displays selected sample spectra Test type Nucleic Acid Selected pot J daDMA aM n Patsa dC Sampia Infcematon dale 480 opal Det 126 2085 IL 25 an Iw Ug rano CLG 15 4T Features include Test Type Auto fills in module name Date Auto fills in date and time of report 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 15 3 Section 15 Archived Data and Data Viewer Plots Sets Users may select the maximum number of individual plots up to 20 graphed per page Since a report can hold data for many samples and a graph page is limited to 20 plots additional sample spectra are displayed on new pages Each page is then referred to as a set Legend Positioning the cursor over the legend box will bring up a visual di
7. even layer of PR 1 to the surface of the upper and lower pedestals 3 Wait 30 seconds for the PR 1 to dry 4 Fold a clean dry laboratory wipe into quarters and remove the PR 1 by aggressively rubbing the surface of the upper and lower pedestals until all compound residue is removed Note The black appearance of the removed residue is normal The reconditioning process is complete once the laboratory wipe shows no more black residue To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up As an alternative to using the PR 1 Kit the pedestals may be reconditioned us follows e Fold a clean dry lab wipe over several times to increase its thickness e Press the lab wipe firmly down on the lower pedestal and buff rub very aggressively at least 50 times the lab wipe will rip during this procedure and will have to be refolded several times throughout the procedure The upper pedestal may also be buffed but care should be taken not to put too much force on the upper arm To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up If the warning persists and the user visually confirms that the liquid column is forming contact your local distributor or Technical Support for assistance Saturated Detector The detector is saturated fo
8. AJ 3054 SU 3 87 LES 225 rcp eye ee a er iL SCT Ha ioe eh 1 ron abs Sms Ac hd agful Lo d Ganga rampe w AAD 15 faves in ern 213 D AG GEM GER En En Sa En eee e Sample Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 for RNA ssDNA 33 for single stranded DNA or Other for other nucleic acids The default is DNA 50 If Other is selected the user can select an analysis constant between15 150 When navigating amongst the three general sample types within the Nucleic Acids module the last constant value entered within the Constant 6 1 Section 6 Nucleic Acids sample type will be retained See the Concentration Calculation Beer s Law Appendix for more details on this calculation 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 a
9. and is ready for quantitation of protein samples at startup This module displays the UV spectrum measures the protein s absorbance at 280 nm A280 and calculates the concentration mg ml Like the Nucleic Acid module it automatically switches to the 0 2 mm pathlength at very high concentrations of protein Also analogous to the Nucleic Acid module the Protein A280 module displays and records 10 mm 1 cm equivalent data on the screen and in the archived data file Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension properties in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter the surface tension resulting in difficulty forming adequate columns for measurement The column formation issue can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric 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 occu
10. be used to load a previously saved curve generate a new standard curve or view the current standard curve SHIN LI Protein BCA File Edit Configuration 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 mg ml 1 000 Average absorbance 0 249 Abs Replicates Delete au Selected Le EE 0 254 0 257 Reset Alternatively previously saved standard curves and standard curve concentration series may be loaded using the Standards menu bar drop down options e Measure Standards Up to 5 replicates of each standard will be displayed The software will not allow measurement of samples until a minimum of either a reference and 1 standard or 2 standards are measured Polynomial curve fitting requires more standard points depending on the polynomial degree selected E BCA Protein Standard Curves File Standards Help Print Page Ap us 0 2 L LL
11. center positive 17 3 If none of the troubleshooting steps above solves the problem contact your local distributor or Technical Support for assistance 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 and recognized Error Code 8 P Error Code 8 Error reading or writing to file This might be caused by 1 The current Windows account does not have Read and Write priveleges to the folder C NanoDrop Date and all of its subfolders Contact your PC administrator to give all users Read and Write access to these folders 2 The log files have been removed from the folder C ANanoDrop DateMog files Replace the log files if they have been moved Ifthe log files can not be located reinstall this software 3 The log files described above are setto read only Check the properties on each ag 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 WD 8000 Data or one of its subfolders See your PC administrator to make sure that all users of the opera
12. 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 is listed below 10 1 Section 10 Proteins amp Labels Approx Typical Reproducibility EEN Upper minimum 5 replicates Limit SD mg ml CVz sample range 0 15 5 mg ml 0 15 mg ml STEE sample range 10mg ml 2 5 96 sample range 0 25 4 pmol ul 0 25 sample range gt 4 0 pmol ul 2 5 96 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 Il
13. data columns of interest when exporting data Save the designated path by clicking on the Save amp Exit button before exiting the User Preferences module 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 t 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 Utilities and Diagnostics This module is used to help troubleshoot operational problems with the i
14. e 800 MHz or higher processor e CD ROM drive e 128 MB or more of RAM e 100 MB of free hard disk space e Open USB port the instrument can only be connected via the USB port e Microsoft Excel or other spreadsheet program to manipulate archived data optional Software Installation WARNING The system software must be loaded onto the PC before the USB cable is connected Administrator access on the PC is required to install the software When attaching the USB cable please wait at least 30 seconds for the USB devices and internal drivers to be installed and recognized 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 and the Found New Hardware Wizard should start as shown below Windows XP SP2 operating system will ask to allow it to search the internet for the proper software as shown Select No not this time Follow the prompts for automatic installation of the software Welcome to the Found New Hardware Wizard elke val search En cunei e neg on pour comporter on her adesse iri abahon LL re H ek Joxiegfe weg ote lte HERE peer Desiree Gei
15. e Show Report formatted for 1000 samples although the buffer size can be modified 9 2 Section 9 Protein A280 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 9 3 Section 10 Proteins amp Labels 10 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 in the main menu 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 ent
16. 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 using the mass extinction coefficient of 6 7 at 280 nm for a 196 10 mg ml BSA solution IgG reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 13 7 at 280 nm for a 196 10 mg ml IgG solution Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution User entered values for molar extinction coefficient M cm and molecular weight MW in kilo Daltons for their respective protein reference Maximum value for e is 999 X 1000 and maximum value for M W is 9999 X 1000 User entered mass extinction coefficient L gm em for a 10 mg ml 1 solution of the respective reference protein e nm 1 and nm 1 abs current value of the user selectable wavelength cursor and corresponding 10mm equivalent normalized absorbance at the respective wavelength absorbance The wavelength can be set by using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e A280 10 mm Path 10 mm equivalent absorbance at 280 nm for the protein sample measured e A260 280 ratio of sample absorbance at 260 nm and 280 nm
17. range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing 9 1 Unique Screen Features Ene Ede Conbgurmbon Measure Eet ii Dampies Type bep Aw 16 0 Age 1583 Lie E sape AJ 16080 bun enz Ub maim Section 9 Protein A280 7 7 maim 4 65 TE iml OAI mami 24 34 nere mg ml 24 45 mg m 2473 DS mg ml 7459 24 zh MES mg ml 74 Bl LOCI OE e Sample Type There are six sample types options available for purified protein analysis and concentration measurement All of the options can be viewed by clicking the mouse while it is positioned within the Sample Type box The sample type color keyed can be selected by clicking on the preferred option or by scrolling through the selections using the up or down arrow keys located to the left of the sample type box Note Concentrations for all eight samples will be calculated using the same mass extinction coefficient as determined by the Sample Type selection A description of each sample type is given below 1 00 1 00 Sample Type Otherprotein E 196 v Ext Coeff E 1 L gm cm A general reference setting based on a 0 1 1 mg
18. surface 50 times aggressively with a dry lab wipe If column breakage persisits use a larger sample size 1 5 2 ul 3 One or both of the measurement paths is out of calibration If this warning persists and the above remedies do not solve the problem contact NanoDrop Technologies or your distributor 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 2096 of the long path absorbance within a tolerance The most common explanation is that the column is not forming properly due to the pedestal being unconditioned When a pedestal becomes unconditioned sample droplets applied to the bottom pedestal will flatten out and cover the entire pedestal surface rather than bead up Buffers containing detergents and various other reagents may cause the pedestal surfaces to become unconditioned We have noted that routine use of the Bradford reagent may result in difficulty forming columns with 1 ul samples Reconditioning Use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement 17 5 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
19. the sample absorbance according to the following equation Absorbance log Intensitysampie Intensitypiank Thus the measured light intensity of both the sample and of the blank are required to calculate the absorbance at a given wavelength Concentration Calculation Beer s Law General The Beer Lambert equation is used to correlate the calculated absorbance with concentration A E b c Where A is the absorbance represented in absorbance units A E is the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of liter mol cm b is the path length in cm and c is the analyte concentration in moles liter or molarity M Fluorescent Dyes MicroArray Measurement The software uses the general form of the Beer Lambert equation to calculate fluorescent dye concentrations in the MicroArray module The table of extinction coefficients for each dye is below Extinction Measurement Dye Type Coefficient Wavelength liter mol cm nm 150000 250000 Alexa Fluor 488 71000 Alexa Fluor 546 104000 19 1 Section 19 Appendices Alexa Fluor 555 150000 Alexa Fluor 594 73000 Alexa Fluor 647 239000 650 Cy3 5 150000 581 Nucleic Acids For nucleic acid quantification the Beer Lambert equation is modified to use an extinction coefficient with units of ng cm ml Using this extinction coefficient gives a manipulated equation c A eyb Where c is the nucleic acid concentration in ng microl
20. to the 0 2 mm path when the absorbance value first reaches 1 25 for EITHER of the two wavelengths indicated with the two cursors Note It is important to pre select a cursor position for the wavelength of interest before the measurement is taken If the cursors are set for wavelengths with minimal absorbance the 0 2 mm pathlength will not be utilized Note When the 0 2 mm pathlength is utilized the data will be archived and displayed normalized to the 1 0 mm pathlength The feature can be turned 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 Note The wavelength cursors will disappear if the default settings are changed but will reappear once a measurement is complete 8 1 Section 8 UV VIS 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 1000 samples although the buffer size can be modified 8 2 Section 9 Protein A280 9 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
21. xparting Huclaic Acide Levis Sample ID mequined OFF Aulomabe Column Advance OFF Promp User to Close Dita Vir l FF 3 3 Section 3 General Operation Data Exporting In addition to the primary data storage of archive files at c IND 8000 Data users may elect to export their data to an additional location This option can be chosen under the Data Exporting tab by selecting the Automatic Data Export box and then choosing the file path by clicking on the file folder icon under Data Export Folder The archived files may be opened with Excel by using the right click option of the mouse to open the nd8 file Note It is important to save the file with a new name before making changes with Excel r Upar Pra arenees pie Help Preagine A Lahalr Linen ninm Repora To expo sabes eee a eutgmaben hy eter ear B mesurament Se ck s Dita Ex TA am box 1 r dar nis export det eg Ge Ae iy Se i ae ee S Up aren ekin mA Atha angu Lien Eve atio F KAnsdi ile burion Aunpmnar Data Epot 7 On Test Module Dieta Exp Fa ider ngvidccenkien LS i and en nskto op die si det Seber dale export heids and the order that the heids should appi Com te menm selecting penake an item to the desired poston Optnally enable the export of the raw absorbance data range and log interval Export Fields Abs Spectrum Export Disabled on stant ar Eos Cursor obs This box is used to select the particular
22. 0 is at the same as the diluted sample measured on the second spectrophotometer William W Wilfinger Karol Mackey and Piotr Chomczynski Effect of pH and lonic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity BioTechniques 22 474 481 March 1997 Wavelength accuracy of the spectrophotometers Although the absorbance of a nucleic acid at 260nm is generally on a plateau the absorbance curve at 280nm is quite steeply sloped A slight shift in wavelength accuracy will have a large effect on 260 280 ratios For example a 1 nm shift in wavelength accuracy will result in a 0 2 change in the 260 280 ratio Since many spectrophotometers claim a 1 nm accuracy specification it is possible to see as much as a 0 4 difference in the 260 280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are both within wavelength accuracy specification Nucleotide mix in your sample u The five nucleotides that comprise DNA and RNA exhibit widely varying 260 280 ratios The following represent the 260 280 ratios estimated for each nucleotide if measured independently Guanine 1 15 Adenine 4 50 Cytosine 1 51 Uracil 4 00 Thymine 1 47 The resultant 260 280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260 280 ratios for the four nucleotides present It is important to note that the generally accepted ratios of 1 8 and 2 0 for DNA and RNA are ru
23. 00 ug ml up to several thousand micrograms ml ug ml on the NanoDrop 8000 The best linearity is in the 100ug ml 1000ug 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 defined 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 pate 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 Bradfo
24. 10 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 08 Table of Contents VOT VOW EE EE 1 1 Instramenb DescripHOLD E 1 1 lee rc c rU PS 1 1 ADPIICAIONS nts E 1 1 wci gqcie P C I 1 1 TU SOG D 2 1 Computer Requirements sess nennen nnne 2 1 Software Installation EE 2 1 Software Neier 2 2 Registering Your Instrument 2 2 General OPC ration Eee SH 3 1 The Sample Retention Gvstem 3 1 Software Architecture and Features 3 2 User ele 3 3 Utilities and Diagnostics EE 3 4 Aere ef Managemen EE 3 5 Dye Chromophore Editor esses nnns 3 6 Plate ET nem UEM EE 4 1 Selecta Plate SOOO E 4 1 EE 4 2 Sample Status Color Gode oc adeo o esee ede obese c ee odios 4 3 Sample Position Illuminator n 4 3 Plate Review e Ree eo en enon ene ee cine Let On nee eee ee eee eee 4 3 Common Module Functions c ccccccessssseeeeeeeeccesseneeeeeeeeeen
25. 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 Do you want to load a previously saved Standard Curve or do you wantto make new standards measurements Load Standard Curve New Standards Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Measurements T able Double CEK en Arry now o chenga fe eoncentratian or dele neqHicabes Do you wantto 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 12 2 Section 12 Protein Lowr File Edit Configuration Help Load from file Measure Blank View or Measure Standards show Report Follow the steps below to either generate or modify the curv
26. Assay is an alternative method for determining protein concentration based on the widely used and cited Lowry procedure for protein quantitation Like the BCA and Bradford Assays the Modified Lowry Assay requires that a standard curve be generated before unknown protein concentrations can be determined The Modified Lowry procedure involves reaction of protein with cupric sulfate in an alkaline solution resulting in the formation of tetradentate copper protein complexes The Folin Ciocalteu Reagent is effectively reduced in proportion to the chelated copper complexes resulting in a water soluble blue product that is measured at 650 nm and normalized at 405 nm Pre formulated reagents utilized in the assay are available in kit form from numerous manufacturers Follow the respective manufacturer s recommendations for all standards and samples unknowns Sample Volume Requirements The presence of surfactants or detergents in reagents can significantly alter the surface tension resulting in difficulty forming and or maintaining adequate columns for measurement The column formation issue can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the
27. E d a arse H LE a im Marium LI 8 1 iN Fl werden pen H gh E anpe t F 42 An oe burp C a M Gi Gage Si Sweet L Tia Ave J 1 M Hi ge e e S api iD L 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 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 Unique Screen Features e 600nm Absorbance current value of the absorbance at the 41 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 1000
28. J K L M N Module Nucleic Acid 2 ath 10 mm 3 IS Mware 321ll 4 Firmware USEUDU 2 41 3 NO3 5 Sample ID UserID Date hme nul AJD ACH HM HILD Constant Cursor Por Cursor abe 340 raw Mesure 6 Default 11 1 2005 838 AM 0 n D NaN Nat 5n 230 0 D Blank Default 11 1 2005 8 28 AM 351 n07 003 KK 2 12 DI 230 se D Measure B Default 11 1 2005 8 39 AM 43 76 0 00 0 52 1 6 2 43 zu 230 0 35 0 000 Measure 9 Default 11 1 2005 8 40 AM 393 73 7 07 4 27 1 05 2 35 zu 230 J 353 0 000 Measure 10 Default 11 1 2005 8 41 AM 3704 14 74 00 39 04 1 86 2 24 zu 230 33 075 0 030 Measure Data Storage Hierarchy The hierarchy for archive 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 within an application module folder that is within the User folder as shown below Address D CAND 8000 Data Default MicroArray v E Go Folders x Name Size Type Date Modified S 3 ND 8000 Data S MicroArray 2007 03 21 vi 14KB NDSFile 3 21 2007 4 02 PM I C3 Administrator S MicroArray 2007 03 23 v1 18KB NDSFile 3 23 2007 8 57 AM C Custom report Formats 3 CH Default O Cell Culture o 4 Nucleic Acid User Defined Data File Export Location In addition to the primary data storage users may elect to export their data to an additional location This option can be chosen unde
29. NanoDrop 8000 Spectrophotometer V1 1 User s Manual The information in this publication is provided for reference only All information contained in this publication is believed to be correct and complete Thermo Fisher Scientific shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing performance or use of this material All product specifications as well as the information contained in this publication are subject to change without notice This publication may contain or reference information and products protected by copyrights or patents and does not convey any license under our patent rights nor the rights of others We do not assume any liability arising out of any infringements of patents or other rights of third parties We make no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Customers are ultimately responsible for validation of their systems 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 198
30. See Column Breakage in this section for further details e 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 Perform a Blanking Cycle This will confirm that the instrument is working well and that any sample carryover from previous measurements is not a concern To run a blanking cycle perform the following 1 Open the application software module 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 both measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum varies no more than 0 005 A 1mm path 6 Reload the plate list file before meas
31. a Fluor 594 l sample range gt 8 0 pmol ul 2 5 Alexa Fluor 546 sample range 0 42 6 0 pmol ul 0 42 sample range gt 6 0 pmol ul 2 5 7 1 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 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 Arie 3 1 A ape H i mata Ou Deg aba Im ngul aac IC eat ii Fs Dyepesu 11551 pow 4112 35 Lys Aven gu angie rra T ab Eys alul Semple Typa ss 4 13 k Beete fi Albena WW BS i L Dyepmocul 12270 eur L ps 3 5 Anere 9 amie ren T abe pp Si ngul Ste eee LL De pesci 251 200 34 4 Actes VU Sege rm t ats Dre ab seht d anie IL HL Ab DS EM HME A re ame LI D a as ys ab aiL amp T wl d IL Dyepexu 71 er nr JE 1 Sai 1 12 D IL Demi 11104 280 gg 16 0 8OOOOO00009099 O O LH s 1 omm ct mm 927 uy ay sjleooooooooooQ Of p aieo eee eaghl EMOOODODOOOGODOOC
32. a Viewer at a later date Choose this option to export a tab deliminited text file of the displayed Report Table suitable for import into Excel Export Report Table Only Choose this option to export a tab deliminited textfile ofthe displayed Report amp Standards Tables suitable for import into Excel Export Report amp Standards Tables Using the Full Report option will allow the user to use the Data Viewer to reload the report at a later date The saved report may be recalled using the pull down Load Report If using the Load Report feature the report will be displayed with the default column 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 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 Method Automatically populated with data method type Date and Time Automatically populated when report is generated Report Name User defined designation for the current report 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 samp
33. ailable for importing using the Data Viewer function 15 6 Section 16 Calibration Check 16 Calibration Check The Calibration Check is found within the Utilities and Diagnostics module and is accessed through the Main Menu It is used to confirm that both pathlengths are within calibration specifications Utilities amp Diagnos Select Diagnostics or Calibration Check Diagnostics 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 an aqueous potassium dichromate K2Cr2O7 solution CF 1 for use in confirming calibration of NanoDrop 8000 Spectrophotometers Preparation e Ensure the measurement pedestals are clean and that a 1 5 ul water sample beads up on each of the lower pedestals Follow the cleaning and reconditioning steps below if the water flattens out and covers the pedestal 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 from each upper and lower pedestal with a clean lab wipe 4 Open the vial containing PR 1 and use the applicator provided in the kit to remove a pin head sized amount of the compound Apply a very thin even layer of PR 1 to the surface of the upper and lower pedestals Wait 30 seconds for the PR 1 to dry 7 Folda clean dry laboratory wipe into quarters and remove the PR 1 by a
34. 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 plate 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 1 3 12 j ECKE i ECKE c EE D C E C reeooo G ui eeooo Sample Position Illuminator This lighted guide allows the user to visualize the row of a standard 96 well micro titer plate that is to be sampled for measurement The Sample Position Illuminator corresponds to the sample status color code displayed on the software screen and the pattern of illumination is determined by plate configuration at set up If measurement limits have been defined wells that are out of the defined range will flash on the Sample Position Illuminator immediately after measurement These positions will continue flashing until measurement of the current plate sample s
35. ar Change the order by selecting and dragging a column header string to the desired position Report columns Well Sample ID Date Show All Time ngul jos A280 260 280 260 230 Constant Cursor Pos Cursor abs 340 raw Measurement Type v Selecting OK will return the user to the Report page displaying only the columns of interest Additional options include Sort Allows users to sort data by column example by date or sample name and by either ascending or descending order Save Report Format Saves the current report format as an 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 Load Report Format Allows saved report formats to be loaded either before or after data is imported Print report Will print out only the Report page by default Users may choose whether or not to print out the standards or plots pages by selecting these options under the Configuration drop down on the tool bar Save Report and Load Report There are several options for this feature as seen in the following window 15 5 Section 15 Archived Data and Data Viewer Save Report As Choose how to save the report Choose this option to be able to reload the REISEN repart into the Dat
36. asuring aqueous nucleic acid samples However if you are unsure about the surface tension properties of your sample or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Measurement Concentration Range The NanoDrop 8000 Spectrophotometer will accurately measure dsDNA samples up to 3700 ng ul without dilution To do this the instrument detects the high concentration and automatically utilizes the 0 2mm pathlength to calculate the absorbance The table below lists the concentration range and typical reproducibility for nucleic acid measurements on the NanoDrop 8000 Detection Approx Typical Reproducibility Limit Upper Limit minimum 96 replicates ng ul ng ul SD ng ul CV 3700 ng ul dsDNA 25 3000 RNA sample range 2 5 100 ng ul 2 5 ng ul 2400 ssDNA sample range gt 100 ng ul 2 5 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be set for nucleic acid measurements as defined by the absorbance at 260 nm These limits cannot be set as a default and must be defined each time the applicati
37. ate file folder enabling the user to load a pre configured plate file Plate files may be created using either Notepad or Excel When creating a file enter the sample names in column 1 and the number of replicates desired for each sample in column 2 The file should be saved as a txt file at C ND 8000 data Plate Files In addition individual bar coded samples may be entered scanned into the text file Note If a plate 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 pre configured plate file it is crucial for a user to know whether the data is arranged by rows or in columns to ensure correct sample identification on the report Right clicking on the specific file from the Select plate file to load window will allow the user to open the file with a program such as Excel or Notepad to confirm data arrangement Look rr gt Plate Hes EN i abc 12 3 eet ali t 3 24 06 1 TN ci rir Right clicking an a file from this window will allow the user to open the file with another program to canfim data arrangement e Define Plate File Format This option allows the user to define the data format of a saved plate file The user is first prompted to select a pre existing plate file from the Plate file folder Once a plate file has been selected the Define Plate File Format window will appear This window enable
38. 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 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 ca
39. 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 Do you want to load a previously saved Standard Curve or do you wantto make new standards measurements Load Standard Curve New Standards Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Measurements Table Double Click Cu sey row Mo Change the concentration or dlii replicates Acton I A Paterance Ju Do you wantto load the mute Acive EB G Stondead1 1 000 concentrations and curve type for Active C Stnded 2000 o generating a standard curve from a previously saved standard curve file 4000 4 500 Clicking on the Yes button will allow the user to import just the Standard series without the respective measured values See image on the right above This option is very useful when running a routine series of standards 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
40. d can be caused by insufficient light intensity reaching the spectrometer If you suspect that this is occurring contact your local distributor or Technical Support for assistance Sample Accuracy and Reproducibility If you are 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 e Make sure the sample surfaces are clean before starting the software module A dirty sample pedestal on startup can cause erroneous absorbance readings even negative values and signal saturation It is always a good practice to clean the sample surfaces with de ionized water to remove any dried sample that might be present Note Do not use a squirt bottle to apply de ionized water e Use a 1 5 2 ul sample size Very strange results can occur when the liquid sample column is not completely formed during a measurement While making a measurement visually confirm that the liquid column is formed If necessary try 1 5 2 ul samples to ensure the column is formed Also proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form 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
41. d Curve or do you wantto make new standards measurements Load Standard Curve New Standards Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Measurements Table EE Aube Clock cun n niw k zrama h concentratian or delete replicates E apotarg ri ELE Bi Abs Ab Abs 5 1006 4 500 E Do you wantto load the concentrations and curve type for generating a standard curve from a previously saved standard curve file nectve EE 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 13 2 Section 13 Protein Bradford File Edit Configuration ESCRIBEN 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 sta
42. d be very nearly zero absorbance All spectra are referenced off of this zero 6 2 a s T 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 the fluorescent dye allowing detection at dye concentrations as low as 0 2 picomoles per microliter Fluorescent Dye Selection There are currently ten 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 96 corrections will be automatically utilized for measurement and concentration calculation The default setting is Cy3 Note Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes Dye Chromophore List Editor Dye Chromophore List nm 250 nm 550 Below 650 Delete Alexa Fluor 545 Selected Alexa Fluor 555 i m Alexa Fluor 594 Edit Alexa Fluor 647 selected Alexa Fluor 660 v Note predefined dyes are indicated
43. e MS Sans Serif font 8 point To check that the system font is set to the proper selection 1 Open the Display Properties by right clicking on the desktop and select Properties gt Appearance Additional step 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 pr
44. e 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 5 mg ml 2 000 Average absorbance 0 151 Abs Replicates Delete 0 151 Selected a occ 0 151 0 153 Reset 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 Lowry Standard Curves Eile Standards Help Print Page 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 us
45. e m a 3 ZI Ai Semple 1 pe 1 abe 5425 A260 5 425 P Samples IO tange 17 EE AMO 2 ung 260 290 1 81 2e230 2 34 Expanded Sample Spectrum View The user may display an expanded view of a single spectrum by clicking on the spectrum of interest This view will display one movable cursor with the respective nm and absorbance reported in the boxes at the bottom of the screen Note Data is archived as the measurement is made Changing the cursor position after the data is archived will not change the data files Moving the cursor via the expanded view will not change the selected wavelength positions on the main acquisition page 5 2 Section 5 Common Module Functions 1 gu ua H BU an E O60 r1 050 5 t di SZ om 120 Print Window A Print dialogue can be initiated from the File pull down menu or by typing Ctrl P 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 Standard Curves A standard curve is required every time the BCA Lowry and Bradford assay are run Although curves can be saved and reloaded in the NanoDrop 8000 Spectrophotometer software it is recomme
46. e 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 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 Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng ul dsDNA without dilution Fluorescent dye labeling density of nucleic acid microarray samples Purified protein analysis A280 nm up to 100 mg ml BSA Expanded spectrum measurement and quantitation of fluorescent dye labeled proteins conjugates and metalloproteins e Bradford Assay analysis of protein e BCA Assay analysis of protein e Lowry Assay analysis of protein e Cell density measurements e General UV Vis spectrophotometry 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 XP or 2000 operating system Windows Vista has also been tested successfully with the software The operating software is not compatible with Windows NT 95 98 or ME
47. easured 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 pate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing BCA Assay Sample Preparation Follow the manufacturer s protocol for the assay including recommended incubation times and temperature In addition to the kit reagents protein standards BSA for generating a standard curve for the BCA method are often provided by the manufacturer Use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the NanoDrop 8000 can measure higher protein concentrations than a cuvette based spectrophotometer you may need to supply your own protein standards at higher concentrations than routinely provided by the manufacturer 11 1 Section 11 Protein BCA Unique Screen Features C ACA Proteia File Edd Cenhgurabon Standards Help Hlank Recording Show Hegpce User Dalaull Dale Tame 3 31 2007 12 107 FR L Exil Fisio ID emt Meha nye DLAME Mossuremant mr fete ei Al Sanet meia OU ATO NN saint ampie if ASE MaN HoH
48. ection must be at the level of user or higher may not select an application or method folder within a user in this activity box 15 2 Section 15 Archived Data and Data Viewer Directory tree Used to select specific data to be imported Clicking on the square to the left of each file name will provide further detail to each level Users may choose to select either individual samples within a file or the entire file All import selections must be of the same application or method type 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 Search Function allows the user to locate specific data by searching through sample ID names Sample Information and Spectrum Are populated with the information associated with the most recently highlighted sample Import and Return Uses selected sample data to populate Plots and Reports windows Note Holding down the shift or control PC function keys will allow the user to select multiple samples and or files for importing The keys can also be used to deselect multiple samples See example below par onder j Directory Tree i S I l WE Oh Kat do vo inpar n demciory vus LL Cas abe 1325 randi See corte Launch Archon ds Lonowrier it Ce dcr M make Fi Cose den zT Mer e P E 1 Meanuerraerg Type Menus Le i Archie Fil Converter T Selecied Samples eg po fece
49. eeeeeeeeseaaaeeeeeeeeees 14 1 Cell Suspension Concentrations esses 14 2 Archived Data and Data Viewer eceeeeeee eene 15 1 Aene Fle Creato EE 15 1 RRE 15 2 Tale d Le 15 2 POSIP AOO den MP En 15 3 Standards P ACC acess Sets ensi cat ate casa otras ede dee ecco ER cda aed Ies 15 6 Calibration Check E 16 1 EEGEN Se eege AP 16 1 a 6 61 B f EE 16 2 er 16 2 Additional aire gn 1 Ed EE 16 2 TYOUDIESNOOUING E 17 1 Jett din Lal NOL e e WEE 17 1 So ssee nz q E T T T UU 17 2 Beien c RE 17 4 Beigl 17 4 Other Software Error Messages i Gerpes ein sia edegi iaia 17 4 Liquid Column Brezakage 17 5 reell iere NE NU m EL 17 5 saturated BI io e rec 17 6 Jasuda SOS CII E ee 17 6 Sample Accuracy and Reproducibility cccccccssssseeeeeeeeeeeeeeeeeeeeees 17 7 SE een 17 7 Technical Tee e istinc nett eaten ee a cei peace eat tk atts 17 8 Maintenance and Warranty c ccccccsessessseseeesesseeseeneeeeeeneeseesseseneeneenes 18 1 CC AIAN mei M UE operc 18 1 CANT AMON ep T 18 1 VV all gn EE 18 1 elen Ve 19 1 instrument eer e e 19 1 Blanking and Absorbance Calculations nnnssnnsnnnnneseeeenrnneneenrrnnnennee 19 1 Concentration Calculation Beers Law ccccccceeessseeeeeeeeeeeeseeeeeeeeees 19 1 Solvent COM Pa IDI Y mm 19 2 Decontamination of Measurement A Optical Surfaces 19 2 Section 1 Overview 1 Overview Instrument De
50. efore releasing the pipettor s dispensing mechanism Visually verify all samples are correctly transferred to their respective pedestals 2 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 3 When the measurement is complete open the sampling arm and wipe the samples from both the upper and lower pedestals using a soft laboratory wipe Cleaning the Sample Retention System Wiping the sample from both the upper and lower pedestals as shown above upon completion of each sample measurement is usually sufficient to prevent sample carryover and avoid residue buildup Although generally not necessary 2 ul water aliquots can be used to clean the measurement surfaces after particularly high concentration samples to ensure no residual sample is retained on either pedestal After measuring a large number of samples however it is recommended that the areas around the upper and lower pedestals be cleaned thoroughly 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 de ionized water Decontamination of Measurement Pedestals If decontamination is necessary a sanitizing solution such as a 0 596 solution of sodium hypochlorite 1 10 dilution of common comme
51. er optic cable Decontamination of Measurement amp Optical Surfaces If decontamination is necessary a sanitizing solution such as a 0 596 solution of sodium hypochlorite 1 10 dilution of common commercial bleach 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 19 2
52. ered dyes Dye Chromophore List Editor Dye Chromophore List 260 nm 280 nm rees Below Delete Alexa Fluor 546 Selected Alexa Fluor 555 F 0 04 0 08 Alexa Fluor 594 E dit Alexa Fluor 647 selected Alexa Fluor 660 wv Note predefined des are indicated with a diamond and cannot be modified Sample Volume Requirements Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter the surface tension resulting in difficulty forming and or maintaining adequate columns for measurement The column formation issue can be 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 oolutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not from properly lf 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
53. et 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 4 3 Section 4 Plate Set up 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 Pate Derups we Cl t 2 J 4 7 B c2 0 ost ven ans 03 4583 ep 4595 ep 4588 25 Jeng 4609 del eat POG 455 CH SIS 4617 4611 4606 69 4588 Et 4577 E3 H eng 4583 en DER i 4522 4530 ul E bb 3 bI EI I bah Blt 4613 4538 au 4570 eat en er ei ep ez 4583 0G 03 4607 ep 75 eu er W en ven ei 475 enn et GS ep i 4614 4622 4603 4615 98 B SAS 4615 2423 4619 4617 463g H Seb nn 469 RR 5 4 59 amp DB 456 aS 4S we 2dESZ Call Colar Cada Siok o any cell In eolect de select Thai Ce for topeni measuemants vehas midi briri Bei cuina leeis Lat age misde n s amp elected Deet green outs
54. features and options for the Report page can be found in the section on the Archived Data and Data Viewer Plate ID The Plate ID will be automatically populated if a plate file is imported Alternately the user may manually type in a Plate ID or other identifying descriptor The Sample Position Illuminator allows the user to visualize the row of a standard 96 well micro titer plate that is to be sampled for measurement This lighted guide corresponds to the sample status color code displayed on the software screen and the pattern of illumination is determined by plate configuration at set up Status The Status button will turn green during a measurement cycle 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 Indicates number of replicates Indicates actual number of replicate specified inthe plate set up measurements made Nucleic Acid Eve Edit Configuration Help 000000000 Measure Bienk Rebiank Recording Show Feron user Default DofeTime 3 23 2007 11 40 AM Plate ID plete 1 All Active Ono nmi 250 j Mensureme plete Activ
55. g the Measure button F1 The result should be 8 spectra with relatively flat baselines near zero e Wipe the blank from both measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum varies no more than 0 005 A 1mm path Reload the plate list file before measuring samples if necessary See Blanking and Absorbance Calculations in the appendix for more information on blanking and absorbance calculations 5 1 Section 5 Common Module Functions 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 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 File Edit Configuration tandard Help 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
56. ggressively 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 o o0 e Enter the Target Absorbance in the appropriate box labeled Target absorbance 350 nm 1 mm pathlength The target absorbance is between 0 710 and 0 760 but the actual value can be found on the CF 1 ampoule label and is lot specific r BP BUCU Calibration Check Fie Help Etank Fr Scroen Raset Sg LDetaut Lie Tire TAM 133 FM Target abs 3i350nm 1 0 me pet 0 740 KRap cees 0 Mex Absorbance 100 2 Display Fedesisl Mugasuramgri nmi 3540 Comechonnm EU nissadunl Facdersal rechte C Enter Target Absorbance Picasa enter the Target Abs vs the CF 1 ampoule label This velud rege Ween AD for operation of Calibration Check So 0 350 400 450 500 550 600 B50 700 750 MWenhiength nm 1 03 COM D SA e Before opening an ampoule of CF 1 Calibration Fluid shake vigorously to thoroughly mix solution Ensure all liquid is collected in the bottom portion of the ampoule e Carefully break the neck of the ampoule to open the CF 1 calibration fluid and aliquot 60 ul into each of the 8 wells of the PCR strip 16 1 Section 16 Calibration Check NOTE CF 1 is supplied in a single use vial and must be used within 30 minutes of opening the vial Prolonged exposure to the environment may cause a significant concentration change P
57. he 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 plate set up for more information about the Sample Position Illuminator The entire measurement cycle takes approximately 20 seconds less time if fewer than 8 positions are used Blank F2 Before making a sample measurement a blank must be measured and stored All eight positions are blanked with each blanking command Note 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 lines Blanking Cycle For the most consistent results it is best to begin any measurement session with a blanking cycle e Open the application software module e 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 e Click on the Blank button When the measurement is complete wipe the buffer from all pedestals e Select All Active On and analyze a fresh aliquot of the blanking solution on all pedestals usin
58. he 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 e Dye abs normalized 10 mm equivalent absorbance of selected Dye e Dye uM concentration of dye in sample based upon selected Dye s extinction coefficient See the Concentration Calculation Beer s Law in the Appendix for more details on this calculation Dyes can be selected using the scroll arrows or by highlighting the Dye box e Dye abs nm 1 ratio of the absorbance of the dye to the absorbance at the user selected wavelength nm 1 e mg ml concentration of protein in the sample calculated using the absorbance at 280 nm minus the absorbance at 340 nm i e normalized at 340 nm Note The user may elect whether or not to use the bichromatic normalization of the 280 nm absorbance value to the absorbance value at 340 nm See the Concentration Calculation Beer s Law Appendix for more details on this calculation e Show Report formatted for 1000 samples although the buffer size can be modified 10 2 Section 10 Proteins amp Labels Baseline Ty
59. ide lis 4 salacind bepenm Soalocheg Chou eeschte 4 4 Section 5 Common Module Functions 5 Common Module Functions Module Startup When a software module is opened the first message seen will indicate that the instrument motors are initializing After that quick step is complete the next message will appear Initialize ND 8000 Ensure sample pedestals are clean and then load water samples After loading water samples click OK to initialize instrument 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 Module Functions File Edit Corfiguretion Help AE Gank ennen Show Repor Lk Defaut CaieTime Weayedo 10 19AM Ext Plate IC nm1 60 Make new BLANK Measurement Set 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 t
60. ife scientist s needs It includes the following application modules e Nucleic Acid concentration and purity of nucleic acid e MicroArray dye incorporation concentration and purity of nucleic acid e UV Vis general UV Vis measurements e Cell Cultures absorbance light scattering measurement of suspended microbial cells e Protein A280 concentration and purity of purified protein e Proteins amp Labels concentration of dye labeled proteins conjugates and metalloproteins e Protein BCA protein concentration using the BCA assay e Protein Bradford protein concentration using the Bradford assay e Protein Lowry protein concentration using the Modified Lowry assay User Preferences Each user has the option to configure a number of settings in the various application modules 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 Viewer to close when closing out of an acquisition module Uer Pirelorences File Hel Laser Diesault Micronrre Proteins amp Labels Frosin ASHE General Setings Reports Dwin
61. ing 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 12 3 4 E43 PM Hebra ta Sample Asean V T 2 C614 0 ee ER Oe Ee Modified Lowry Standard Curve 0 2 4 0 mg ml O0E 0 5000E 3 Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module 12 4 Section 13 Protein Bradford 13 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 Vol
62. ing software and overwrite the existing copy when prompted A new copy of the passwords log file should appear in the CAND 8000 Data Log Files folder Error 9003 This error indicates that the monitor resolution is below the 1024x768 required Check the computer settings Be sure that the Start menu tool bar is set to the bottom and not along the side Low Detector Bias This occurs when the software has identified a problem with the detector Contact your local distributor or Technical Support if you encounter this error Driver X Configuration Failed You Must Manually Edit the Registry This error message or others with similar wording occurs when attempting to install the operating software on a computer running Windows 2000 or XP It occurs because the user does not have the necessary authorization to install the software Contact your system administrator if this occurs Insufficient Memory This error message or others with similar wording occurs when attempting to install the operating software on a computer that does not have at least 100 MB of free hard disk space Liquid Column Breakage Warning The ratio between the long path and short path absorbance is out of tolerance This might be caused by 1 An air bubble was entrained in the sample Try measuring 2 The liquid column is not Forming can be confirmed visually If this is occurring try cleaning both measurement pedestals with water and then wiping each
63. ions 1 2 ul It is best to use a precision pipettor 0 2 ul with 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 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 Sample Carryover Prevention of sample being retained on the NanoDrop 8000 Spectrophotometer s measurement pedestals is easily addressed Simple wiping of the upper and lower measurement pedestal with a dry 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
64. ipettor 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 Unique Screen Features e Automatic Path Selection a user selectable feature unique to this module If selected the software will automatically switch from using the 1 0 mm pathlength
65. irm proper USB communication Start gt Programs gt NanoDrop gt Utilities gt USBView If USBView is not installed on your PC you can download it from the Downloads section of our website 10 Click on the Device Connected see example below If more than one USB device is connected view each of them Three of the connected devices should display ID vendor numbers of 0x1B60 with ID products numbers of 0x0003 0x0004 and 0x0005 If all three are present the USB function of the instrument should be OK If the idVendor and idProduct are different than indicated above or if no USB device is present in the list continue to step 11 12 4 E USB View Fie Options Help Intelfr 82801DB DBM USB Un Device Descriptor SB RootHub 0x1020 0x00 Port1 NoDeviceConne ae 0x00 Port2 NoDeviceConne bDeviceProtocol 0x00 Intel r 82801DB DBM USB Un P i 5 0x40 RootHub Fort1 DeviceConnecte Port2 NoDeviceConne Intel r 82801DB DBM USB Un RootHub Port1 NoDeviceConne bNumConfigurations 0x01 Port2 NoDeviceConne ConnectionStatus DeviceConnected Inter 82801DB DBM USB 2 0 Current Config Value 0x01 RootHub Device Bus Speed Full Port NoDeviceConne Device Address 0x02 Port2 NoDeviceConne Open Pipes Port3 NoDeviceConne Endpoint Descriptor Port4 NoDeviceConne i 0x02 Port5 DeviceConnecte Bulk Port2 DeviceConr binterval Port3 NoDeviceCc y Endpoint De
66. iter A is the absorbance in AU e is the wavelength dependent extinction coefficient in ng cm microliter and b is the path length in cm The generally accepted extinction coefficients for nucleic acids are e Double stranded DNA 50 ng cm ul e Single stranded DNA 33 ng cm ul e RNA 40 ng cm ul For the NanoDrop 8000 Spectrophotometer path lengths of 1 0 mm and 0 2 mm are used compared to a standard spectrophotometer using a 10 0 mm path Thus the NanoDrop 8000 Spectrophotometer is capable of measuring samples that are 50 times more concentrated than can be measured in a standard spectrophotometer Note Absorbance data shown in archive files are represented as displayed on the software screen For Nucleic Acid Protein A280 and Proteins and Labels modules data are normalized to a 1 0 cm 10 0 mm path For MicroArray UV Vis Protein BCA Protein Bradford Protein Lowry and Cell Culture modules the data are normalized to a 0 1 cm 1 0 mm path Solvent Compatibility The NanoDrop 8000 Spectrophotometer is compatible with most solvents typically used in life science laboratories These include methanol ethanol n propanol isopropanol butanol acetone ether chloroform carbon tetrachloride DMSO DMF acetonitrile THF toluene hexane benzene sodium hydroxide sodium hypochlorite bleach dilute HCI dilute HNOS dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fib
67. les 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 Leninger A L Biochemistry SH 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 Serial Number of the instrument The number can be found on the bottom of the unit 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 17 8 Section 17 Troubleshooting Application Module Screen Captures Screen captures of the actual spectrum as seen on your PC are of great use in diagnosing problems Making a screen capture is quite easy When in an application module press Alt Print Screen This copies the highlighted screen window to the PC s clipboard Next paste this screen capture into MS Word MS Paint this program usually comes standard with the PC and can usually be found in the Start gt Accessories menu or other graphics programs Save this as a jpg o
68. les per report may be modified by highlighting the box and typing in the desired number Choosing Ignore from the drop down will allow the user to include an unlimited number of samples in a report 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 Plots Report Standards Test type Bradford 10 27 2005 1 17 Standards Sample Curve Ref Ref Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 ID Type conc Abs conc Ahs conc Abs conc Abs conc Reference interp 0 00 0 029 NaN NaN NaN NaN NaN NaN NaN Standard 1 interp 0 00 0 029 100 00 0 047 NaN NaN NaN NaN NaN Standard 2 Interp 0 00 0 029 100 00 0 047 1000 00 0 099 NaN NaN NaN Standard 3 Interp 0 00 0 029 100 00 0 047 1000 00 0 108 2000 00 0 073 NaN Opening Archived Data with Spreadsheet Programs The archived files are in tab delimited format and can be opened in Microsoft Excel or an equivalent spreadsheet program To open these reports go to the C ND 8000 Data Reports folder and right click on the file of interest Note Save and rename files before making any changes if opened with Excel type of programs to ensure that the original archived data is av
69. luminator will stop flashing Unique Screen Features Active E i A Al Smpn 1 mola 2790 Dpeaba SIIN tampleiD Treg Io C uA Dweud 2614 Dye going aha 1908 zu PC ed Actee A l Hi Sepkil i mn dn 254 Dee 53505 mml Sample Type IgG we Besclslff maia A A 2 DyeuM 1647 Dye aire aha H gan Acpeg 8 1 A Cl Sener 1 mabe 2305 wea 51 23 mami Sample D zmg milga Dead 1553 Ope aime abs TAT 2 03 Bezelea Type 250 nm E Apte E L U DI Sample T 1 mila Jae Oy ate SIT mrp Sere D Faggi kan th DpeuM JERE Dye ah abe TU zn Adwe W d ET Seet 1 midn 2863 Dn 51518 wait r snl a FETT nens 157 L T Semple ID ergy rel haa TX EN DyeuM AER Dpe abot aha Hba i d i Lal 12 ale fees 8 d Fl Sample mise 234 Dyeae Han eat Bla SwweiD Zmomilg ho A IU eT Dye singe obs 18 2 1 96 cue olgoooooooooono Segel i d GI reen 1 mis 2900 Dye 5020 mim rleoooOoOoOOOOOO 0 moms PA Dm 3e Dye rie aba 817 E i Ht Sano H 1 Ten ga 2753 Dye n 52445 mml u le2oooooooooo e Sample Type The same six sample types options listed in the Protein A280 section are available for purified Proteins amp Labels analysis and concentration measurement All of the options can be viewed by clicking the mouse while it is positioned within 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 t
70. n Start Report must be selected in order to access the Data Viewer via Show Report Tool Bar Features common to all three pages include File 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 Configuration Options controlled by this tool bar function include Auto Scale Include graph in printout and Include standards in printout Data Includes options to import data rename samples and delete sample data Note After deleting all samples it is important to exit out of the Data Viewer module and re enter if importing data for a different application type Reports This tool bar function allows the user to select columns of interest to be included in a report See following section on Reports Page for details on additional drop down box options s Print Window The current Plot Report or Standards screen may be printed by selecting Print Window or CTL P Save Window Saves files as JPGs 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 Import folder Used to select folder where data is to be imported from Folder sel
71. n 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 he 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 3 5 Section 3 General Operation Note The administrator using the Options or the Modify User entries in the Account Management module establishes whether individual user passwords will expire and if so after how many days Maximum Password Attempts li Minimum Password Length Password Expiration enabled LI Default Expiration Days aen a 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 WD 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 Dye Chrom
72. nation 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 Routine use of ethanol or isopropanol for cleaning is not recommended 14 2 Section 15 Archived Data and Data Viewer 15 Archived Data and Data Viewer Sample data from all application modules is automatically stored in archive files and can be opened by either the integrated Data Viewer software program or spreadsheet programs such as MS Excel Archive File Creation Every time an application module is started an application specific archive file is created for the user that is logged in All measurements made by the user in that application module for a given calendar day are stored in a single archive file These files bear the name of the respective application module with the date appended For example an archived file entitled Nucleic Acid 2007 03 21 nd8 corresponds to Nucleic Acid data from the s
73. nd 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 2 Check that the power supply is connected to the instrument Confirm that the instrument is getting power by observing that light can be seen through the USB opening on the rear of the instrument 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 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 NanoDrop Devices If an unknown device a yellow exclamation point or a question mark appears next to one of three expected NanoDrop devices manually uninstall the device by right clicking and selecting Uninstall from the options displayed Disconnect the power supply from the instrument first and then disconnect the USB cable from the NanoDrop 8000 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 f
74. ndard or double click any where in the row of a particular standard to bring up the Edit Standard dialog box to enter in the concentrations for each standard This box is also used to delete a single absorbance replicate value or reset the entire standard Edit one standard Standard Standard 4 ug ml 1000 0 Average absorbance 0 124 Abs Replicates Delete a ulead 0 123 acc 0 129 0 123 Reset 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 Bradlord Standard Curves 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 mus
75. nded that the user follow manufacturers quidelines 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 assay reagent only no protein and a single replicate of one standard There is no set order in which standards must be run The multi point standard curve generator allows a maximum of 5 replicates for each of 7 different standards The following box will appear after the module initialization is complete Choose Standards Source Do you want to load a previously saved Standard Curve or do you wantto make new standards measurements Load Standard Curve New Standards Measurements The user may load a previously saved standard curve or generate a new curve Selecting the New Standards Measurements button will bring up the dialogue box on the left below Srandard mid jaa Aes Abs Abs 2 Abs 3 Abs d Abs 5 Do you want to load the concentrations and curve type for T D Standard 3 4 000 generating a standard curve froma previously saved standard curve file E Standarda 4 500 F Sandang 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 The Standards menu drop down may also be used to l
76. nna QUU AUS Ha mg ml E ampla iD SES Mak Hoh j ooooooOocOOOO T EA P 8 Art a 1 te in S D a i j d OOOOOOOO0OoO0O0 we i eqn B mle BON AUS H i een CH CH C e CH Ce CH CH CH CH CH CH Samed D 3 He Hall Ha Actio 7 1 H1 Sexe Hi D ml Sa DON BAG MaM maimik AP HH Hoh CC vu TE Sanpete ID e A 650nm the Cu complex s absorbance at 650 nm for the 1 mm pathlength e nm 1 and 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 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 e Show Report formatted for 1000 samples although the buffer size can be modified 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
77. nstrument For more information on using this module refer to the Troubleshooting section of this manual 3 4 Section 3 General Operation Account Management The Account Management module provides options for directing where specific data files are archived allowing users to segregate their data into personal folders The Account Management module is accessible to the administrator only User Access Manager Actions All Users User ID Full Name Level Active Locked Expired Expires Default user 0 Active NotLocked NotExpired Never Administrator Administrator NotLocked Not Expired Never Account Types There are three types of user accounts Level 10 this is the highest security setting and all level 10 users can add new users modify a user delete a user and set password options At the time of software installation the only level 10 account is Administrator whose initial password is nanodrop Itis 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
78. o make a sample measurement e Auto Advance Columns If selected there is a 5 second delay before the displayed spectra clear and the status code and Sample Position Illuminator see below for description of these features advance to the next column for sampling This gives the user the option of canceling auto advance for that column Auto advance will restart when the next column is measured This feature can be set as a default function under the User Preferences application Auto Column Advance Automatic column advance will occur in 4 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 4 2 Section 4 Plate Set up e Measurement Limits Allows the user to set minimum and maximum concentration limits for samples to be measured Additional information about setting measurement limits is included in each application module section Enter Measurement Limits Enter the Minimum and Maximum Concentration Sample measurements that fall outside this range will be highlighted in displays Min Concentration Inf Max Concentration m S e Show Plate Summary Allows the user to review the results of the plate at any time during the measurement session Once the entire plate has been read this feature will
79. oad a previously saved curve generate a new standard curve or view the current standard curve See the respective protein assay section for additional details User s Manual The User s Manual is accessible from the Main Menu and from the Help menu in all of the application modules It can also be accessed by selecting Start gt Programs gt NanoDrop gt ND 8000 version 5 3 Section 5 Common Module Functions 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 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 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 5 4 Section 6 Nucleic Acids 6 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 me
80. oftware 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 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 L Microsoft Excel Nucleic Acid 7005 11 01 v3 2 ndj Read Only i59 f e cdt Yew Insert forme Took D s Window ACTI Hel Adobe FOr eRe FE Ie weep IET An 10 B FEULEN E e xl ie Al H f Module E Forms geb E F G In
81. on module is opened Nucleic acid measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the plate summary has been reviewed samples of interest marked for repeat and the window closed the Sample Position Illuminator will stop flashing Unique Screen Features Die Edi Coniquresdon j Help Measure Slank Perblonk Berck Stim Report Lee Diaidh Deme fure 200 11 40 AM Exin Pate ID plane T nmi 250 AAcasurimant eamplehe Acie m W ilis Ai Semen i rel gu 5425 AED 5 425 agul A A Sample temple d X AE ig acoso 10 awa 234 ma ache m 3 TS BJ fees 1 malae 550 AO ES nn ul ms Sample Typo OMS zeiet vue 18 Y ace NUM awa UD og 2 5 E Acie PL 3 Ts CS See 1 eigei E A2 5495 ngni Sarge iD sie 15 A ic Ag 308 emm 10 um 22 KR Acwe Wl 2 df D I Seet 1 mise SS ABS EIS sait i w Sample EI 1 sample 20 DN A38 3062 Savon itt Sars 2 2 aaa m 3 TEP Seet 0 niam a5 AGB 5451 ngul Walk o Smal rampie 21 bb aiii oe 83 ink 2 5 1 EJERE TA ees EE ZC APeoooooooooo Adwe N 8 3 LATIN ET Sehala neam nam AB STE nghil BIgagpoooooOOOOTQOCG ssen ez LENT As 2390 oO 18b eum 237 B7 Aster Di E 1 3X sjleeeoooooocoog ET VN d ue nn Nm d A H
82. on our website 18 1 Section 19 Appendices 19 Appendices Instrument Specifications e Sample Size 1 microliter e Sample Number up to 8 e Path Length 1 mm with auto ranging to 0 2 mm Light Source Xenon flash lamp Detector Type 2048 element linear silicon CCD array Wavelength Range 220 750 nm Wavelength Accuracy 1 nm Wavelength Resolution 3 nm FWHM at Hg 546 nm Absorbance Precision 0 003 absorbance 1mm path Absorbance Accuracy 2 at 0 76 absorbance at 257 nm Absorbance Range 0 02 75 10 mm equivalent absorbance Detection Limit 2 ng microliter dsDNA Maximum Concentration 3700 ng microliter dsDNA Measurement Cycle Time 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 or XP Vista has also been tested successfully with the software 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
83. ophore Editor The Dye Chromophore Editor gives the user the ability to add additional dyes or chromophores to the list of predefined fluorescent dyes available for use with the MicroArray and Proteins amp Labels modules Predefined dye methods are indicated by a diamond and cannot be modified Absorbance contribution at 260nm and 280nm from the respective dye can be corrected by entering the appropriate decimal correction in the respective field when adding a new dye to the list Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes Dye Chromophore List Editor d om Below Dye Chromophore List Alexa Fluor 488 D E Delete Alexa Fluor 546 1 04E 5 E Selected Alexa Fluor 555 1 50E 5 Alexa Fluor 594 7 30E 4 Edit Alexa Fluor 647 2 39E 5 SEE Alexa Fluor 660 1 32E 5 Cy3 5 1 50E 5 e Cy5 5 2 50E 5 v Note predefined dyes are indicated with a diamond and cannot be modified 3 6 Section 4 Plate Set up 4 Plate Set up Selecting a Plate Setup After the instrument has completed the initialization process the following window will automatically display the options for plate setup Select Plate Setup Mode Do you want to load a plate setup file or manually enter the plate setup information Load Plate File Manual Plate setup Define Plate File Format e Load Plate File This option opens the Pl
84. or 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 hound Mow Ha n TP Wirard hound Now Hardware Wizard Wobloome to the Found Now Hardware Wizard winders r resch for cumeni and epdabed rofin Es Tha wizard halpa sou gud piae pr kache ini pas cormpulel on Ee haider risky LD oi on Bob lirics LI pubs V eb e euis peur portati Mee LII Dae Bead ur preacs policy f d gow hedeare case wih ze nn ballation LT AE ra Boppy dick eet df nass Lars Wirek Coesech lo Week Leda le sesch Ho softens CO Yer ge me ony Cy Ter noe and greeny ime connect a deca WA ds ya enit Pe esed I dei hio nor So pe 3 rtt ad Ba tein ducit eredi rl ien nnm Ci irala home ia lest or gpecific location advanced Chek Hes bo omnia Chek heal fa conti gees comm cpa ex Izeg Intro Page Windows XP SP2 Other Windows Operating Systems If the Found New Hardware Wizard appears installation will require two cycles through the Wizard once for the spectrometer and once for the peripheral control devices that are internal to the instrument These devices need to install successfully for the NanoDrop 8000 to operate Restart the operating software If the software works properly you are finished If it does not operate properly continue to step 9 Close the operating software and open the USBView utility to conf
85. or barcode scanner The wall position Increments aulomalically wilh each entry Click on awell to go to that wall E samplos Ski Se T2 DODODODDODDO die elect wall coles replicates to B Lx D E rr T i CX DDDDDDDDDDDD DDDDDIDDODDODD DIDDDDIDDODIODI DIDDDODCODODIOILD DDDDDDPDDODIDD Pind Svo Firmi hrd Lanci REES Toabon AJ Replicotas 1 Sample ID To move to the next well in a column enter the sample name and then click on the Next Well button To select a well in another column highlight the well of interest enter in the sample name and hit the keyboard Enter button e Cancel Closes the window without making any changes Configuration 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 From the Configuration drop down the user has the following options File Edit Beine EUIUN Help Define Plate File Format Load Plate File Manual Plate Setup Plate ID Sample ID Required Auto Advance Columns Prompt Close Data Viewer Measurement Limits show Plate Summary e Sample ID Required If selected the sample ID field must be populated for each sample tested Wells requiring a sample ID will appear red in the sample status color code see below for description of this feature and the following message will appear when the user attempts t
86. otection provided by the supplied power cord and power supply The power supply can remain plugged into the NanoDrop 8000 Spectrophotometer while the instrument is not in use When the instrument is plugged in but not in use the power consumption is 3 W and the flashlamp is not energized Also the instrument does not utilize a power switch or give a visual indication of the operability of the 12V power supply Note It is recommended that the instrument not be positioned in a way that makes it difficult to unplug the power supply from the unit or the wall 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 and all information supplied is completely confidential You can register your instrument on our website 2 2 Section 2 Initial Set up Lock Attachment Port The NanoDrop 8000 is equipped with a lock slot that enables use of a standard locking cable typically used to secure a laptop PC 2 3 Section 3 General Operation 3 General Operation The Sample Retention System The main steps for using the sample retention system are listed below 1 With the sampling arm open position the pipettor using the guide as shown Dispense the samples onto the lower measurement pedestal ensuring the samples touch off contact the lower pedestal Carefully withdraw the pipettor b
87. pe mma QUUD ATE Hal ug mL o0 O000000 O00 HF WaN sla ID L AES Mal a QUOUDUODCOOOOU F e A 595nm protein dye complex s absorbance at 595 nm at the 1mm pathlength e nm 1 and 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 or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations e ug mL the concentration of the sample unknown e Show Report formatted for 1000 samples although the buffer size can be modified 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 Do you want to load a previously saved Standar
88. pe 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 10 3 Section 11 Protein BCA 11 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 CuSO 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
89. pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not from properly If this occurs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found on our website Measurement Concentration Range The Modified Lowry assay concentration range of detection is 0 20 mg ml to 8 0 mg ml BSA on the NanoDrop 8000 By selecting Measurement Limits from the configuration drop down menu minimum and maximum concentration limits can be defined for protein mg ml measured with this method at 650 nm These limits cannot be set as a default and must be defined each time the application module is opened Protein measurements that are outside of the defined range will be indicated by a flashing light on the Sample Position Illuminator The sample concentrations will also appear in red when the plate summary is displayed Once the pate summary has been reviewed samples of interest marked for repeat and the
90. r 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 WD 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 17 9 H MM M ea Gr e E TRE RERERDTERERER 18 Maintenance and Warranty Cleaning The primary maintenance requirement of the NanoDrop 8000 Spectrophotometer is to keep the measurement pedestal surfaces clean Upon completion of each sample measurement wipe the sample from the upper and lower pedestals to prevent sample carryover and avoid residue buildup A final cleaning of all surfaces with de ionized water is also recommended after the user s last measurement Note Do not use a squirt bottle to apply bleach or de ionized water 1 Apply 5 ul of dH20 onto each bottom pedestal 2 Lower the upper pedestal arm to form a liquid column let it sit for approximately 2 3 minutes 3 Wipe away the water form each upper and lower pedestal with a clean lab wipe Note Typically dH50 is sufficient for removal of samples that have dried on the optical pedestals There are a few cases i e dried proteins that may
91. r all modules If Start Report is displayed the accumulating data will still be archived but will not be shown in the active report File Edit Configuration Standards Help Show Report Selecting this button will bring up the Report page Hitting the Exit button at the top right will exit back to the acquisition module The data will continue to populate the report after exiting HD B8O000 Data Viewar vi Els Combeaurghra Dele Riot Help Fins inpar Testtypa Riepart Hama Pspor Full Hode E T Wall E Date pus CS A AZOO ILI 8 ST 2h0 200 260 Frnt Seve and Frim 15 4 Section 15 Archived Data and Data Viewer All data is stored in the archive file at c WD 8000 Data and may be exported to an alternate location To open these reports go to the C IND 8000 Data Reports folder and right click on the file of interest Some key options useful for the Report page are accessible through the Report tool bar drop down ND 8000 Data Viewer vi File Configuration Data Help Configure Report Plots Report Sort Report Testtype Report Full Mode Ignore v Report Name Save Report Format Load Report Format Save Report Load Report Print Report Choosing the Configure Report option brings up the following box Report configuration editor Select the report columns to display and the order thatthe columns should appe
92. r the Data Export Exporting tab in User Preferences on the Main Menu 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 15 1 Section 15 Archived Data and Data Viewer Data Viewer Data Viewer is a versatile data reporting software program incorporated into the operating software that offers the user the ability to customize report structures import stored data and re plot data from previously generated data Using the Data Viewer is the most expedient method to review data This feature may be accessed during measurement sessions from the Show Report function found within each method module It may also be accessed from the Main Menu page A NanoDrop 8000 Spectrophotometer does not need to be connected to the PC to use the Data Viewer module Data Viewer Features The Data Viewer is composed of two or three pages in a tabular form consisting of Plots Reports and Standard Curves where utilized The user may access any page by clicking on the tabs The software opens to the Report page whether accessed through the Main Menu or Show Report Note Recording rather tha
93. r the following channels B This is mostlikely caused by 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 PeELEEEEEEGEE 2 Ho s ao ah alo aio e Ho s dr xe sb sw se zip 3 Detector saturation nucleic acid Detector saturation Bradford measurement measurement 17 6 Section 17 Troubleshooting A spectrum that is very un smooth or ragge
94. rcial 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 3 1 Section 3 General Operation Rapid Reconditioning of the Sample Retention System The Bradford reagent as well as other buffers containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form well with 1ul samples Use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Sample Size Requirements Although sample size is not critical it is essential that the liquid column be formed so that the gap between the upper and lower measurement pedestals is bridged with sample Note It is not necessary to have liquid on all 8 positions to make a measurement Field experience indicates that the following volumes are sufficient to ensure reproducibility e Aqueous solutions of nucleic acids 1 ul e Purified protein 2 ul e Bradford BCA or Lowry assay 2 ul e Microbial cell suspens
95. rd Assay Bradford 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 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 13 1 Section 13 Protein Bradford Unique Screen Features Ele Edt Configurstion Standards Help Blank Racerding ShowRepor User Default Date Time 3 71 2007 12 04 PM Exit Pete 0 AB Actium Cu ami 535 Make nee DLANE Measurement aii frie B 1 A Al Sarpa H malas QUU A750 Had vote seis AS Ha NaN fete amp 1 Di Soe D mmie Don ATO HaM voie Zack iD I ASA Ha MaN Standands uires E 1 Cl Sample pn aa Dn AU HH ug mL e Acie 1 1 DI Sanpe H H nm labs GG AED Ha View Jodale Gierdeegz Sai REESEN Kaes ID A ASA Hai Hah Ache Li t 1 E1 E aigla mma DO AMO HaM point x Saepe ID A A Hai Na 1 Led H BH GL x T Adme t 1 FI SampeH O miel DD ATUS Hah atm sample IL AGIS Ma Mahi Acive B 1 DI Somes miaa Dd ATS Hah tery Samed ID L ANE HoN NaM ge d Actes lp H1 w
96. require a more rigorous cleaning protocol For these cases we recommend that 0 5M HCI with D ul of dH20 to remove any residual HCI 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 Calibration Pathlength Accuracy Calibration Check It is good practice to check the calibration every six months using the CF 8 Calibration Fluid Kit This will verify the pathlength accuracy of the instrument 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 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 Warranty All NanoDrop spectrophotometers and accessories manufactured by Thermo Fisher Scientific are warranted against manufacturing defects in parts and labor for a period of one year Preventive Maintenance as well as additional one two and three year warranty extensions are available Additional information about the various plans may be found
97. rocedure e Using an 8 channel pipettor simultaneously add a 1 5uL aliquot of water to each pedestal position lower the arm and click on the Blank button At the end of the measurement cycle use a lab wipe to remove the water aliquots e Using an 8 channel pipettor simultaneously add a 1 5uL aliquot of CF 1 Fluid to the pedestal and click on the Measure button At the end of the measurement cycle use a lab wipe to remove the CF 1 aliquots from both the upper and lower pedestals e Repeat and measure a total of 5 separate sets of replicates Results After the 5 measurement the Calibration Check Results will be displayed on screen e 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 a JPG image at C ND 8000 Data Calib check e To print a copy of the results for your records click the Print Screen button A JPG of the final results is automatically archived on the hard drive at C IND 8000 Data Calib check f the instrument needs to be recalibrated contact your local distributor or Technical Support for assistance Additional information Cleaning instructions the calibration check procedure as well as a table of calibration check tolerances may be accessed via the menu bar Help drop down feature 16 2 17 Troubleshooting Instrument Not Found No ND 8000 was found This error might appear upon software startup a
98. rs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found on our website Measurement Concentration Range The NanoDrop 8000 Spectrophotometer will accurately measure protein samples up to 100 mg ml BSA without dilution To do this the instrument detects the high concentration and automatically utilizes the 0 2 mm pathlength to calculate the absorbance The table below lists the concentration range and typical reproducibility for purified BSA measurements on the NanoDrop 8000 l Typical Reproducibility Sample Type uoc Approx pper minimum 96 replicates SD mg ml CV sample range 0 15 5 mg ml 0 15 mg ml Purified BSA 0 15 mg ml 100 mg ml sample range gt 5 mg ml 2 596 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
99. s cccccccccssessecceeeeeceeeeeeeeeeeseaeeeseeeesesaaeeeseees 10 2 stilum 10 3 s Protem BCA eT 11 1 Sample Volume Heouremente esses 11 1 Measurement Concentration Range s sssssssssesssrrrrreesrrrrrrrrrresrrrrrrnree 11 1 BCA Assay Sample Preparation ccccccccceccsesseeeeeeeeeeeeeeeeeeeeeessaeneess 11 1 Unique Screen Features iieri ossaa e d a aei 11 2 Making BCA Meaesuremente cccccccccceseeeeeecseeeeeeeeeeeeeeeesaeeeeeessaaeeeees 11 2 12 13 14 15 16 17 18 19 Protein LOWY M 12 1 Sample Volume Heouremente 12 1 Measurement Concentration Range sssssssssseessrrrrrreesrrrrrrrrrrenrrrrrereee 12 1 Modified Lowry Assay Sample Dreparaton neren 12 1 Unique Screen Features nennen nnns 12 2 Making Lowry Measurements n nnenenneesnnenennnnsrrrnsrrrnsrrrnsrrresrrresrrenne 12 2 Protein BIadfoEd uio huie EEN 13 1 Sample Volume Heourement srarirsrireino nirien n ea ea 13 1 Measurement Concentration Hange 13 1 Bradford Assay Sample Preparation ccccccccccccccceecseseeeeeeeeeeeesaeeeees 13 1 Unique Screen Features cccccccccsssseeceeeeeeeeeeeeeeeeeeessaeaeeeeeeeeessaeaeseees 13 2 Making Bradford Protein Measurements nnnennnnennenennneesnneesrnnennrnnne 13 2 GON CUNEO e src ce ae agra a eras ree ee uae ee nr 14 1 Sample Size Requirements ccceeeecceeeeceeeeeeeeeeeeesaees
100. s 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 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 ng ul sample concentration in ng ul based on absorbance at 260 nm and the selected analysis constant See the Concentration Calculation Beer s Law in the appendix for more details on this calculation e Show Report formatted for 1000 samples although the buffer size can be modified Spectrum Normalization The baseline is automatically set to the absorbance value of the sample at 340 nm which shoul
101. s the user to define the data series type rows or columns which column contains the sample ID whether headers are defined in the data set and the number of replicates for each sample Selecting Continue after defining the format will load the plate file Note Once a plate file format has been defined that format will be retained and applied to subsequent plates until the user selects a different format For this reason it is crucial for a user to know whether the data is arranged by rows or in columns to ensure correct sample identification on the report Right clicking on the specific file from the Select plate file to load window will allow the user to open the file with a program such as Excel or Notepad to confirm data arrangement T Define Plate File Format Enter the Eatings for Pomel Plane Files Lee d et Papliestas is checked you must anir the Fiat File column man holds the al Repkecsnes N lait unchecked foi Foeplicatas will be 1 forall sompies m re Pale File ghi File CAD DD DigrelFinin ilr sbch23 2heole tet Show Fiat 1 1 1 eT T Dabs Senes Tyne ln Pies AT AZ 3 ke Sample Column 7 S Use ofReplicaics e Manual Plate Setup 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 4 1 Section 4 Plate Set up Enter sample Is Enter the Sample IDs via the keyboard
102. 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 oolutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form properly If this occurs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found on our website Measurement Concentration Range When using a 20 1 reagent to sample volume dilution the BCA assay concentration range of detection is 0 20 mg ml to 8 0 mg ml on the NanoDrop 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 m
103. samples although the buffer size can be modified Sample Size Requirements Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous samples However if you are unsure about the surface tension properties of your sample or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample Use an 8 channel pipettor when loading multiple samples to minimize evaporation due to delays in sample loading It is recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy 14 1 Section 14 Cell Cultures 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 This makes it possible to directly monitor concentrated cell suspensions Since the entire spectrum is displayed diluted samples exhibiting very low Absorbance at 600 nm can be monitored at lower wavelengths for example 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 Decontami
104. scription The Thermo Scientific NanoDrop 8000 Spectrophotometer is a full spectrum 220 750nm instrument that measures 8 individual 1 ul samples with high accuracy and reproducibility It utilizes the same patented sample retention technology utilized on the NanoDrop 1000 Spectrophotometer and the NanoDrop 3300 Fluorospectrometer The surface retention system holds the sample in place eliminating the need for cumbersome cuvettes and other sample containment devices Clean up is accomplished in seconds In addition the NanoDrop 8000 has the capability to measure highly concentrated samples without dilution 50X higher concentration than the samples measured by a standard cuvette spectrophotometer Operation Up to eight 1 ul samples are pipetted onto the sample pedestal using a low volume multi channel pipettor Each position is actually the end of a fiber optic cable the receiving fibers A second set of fiber optic cables the source fibers are brought into contact with the liquid samples causing the liquid to bridge the gaps between the fiber optic ends The pathlengths are automatically controlled to 1mm and 0 2 mm paths Readings are acquired through sequential measurement across the 8 positions A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light that passes through the samples The instrument is controlled by PC based software and the data is logged in an archive fil
105. scriptor lt gt Devices Connected 4 11 Install the instrument on another PC to rule out a faulty USB hub port on the original PC Run USBView on the oe PC 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 If this does not solve the problem refer to the Troubleshooting section of the User s Manual available from Main Menu This error occurs whenever the USB connection is disrupted while operating a software module 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 selecting Retry
106. splay matching the sample name to a plot color The user is not able to select or highlight a sample from the legend Sample information Automatically populates with data associated with selected sample Data displayed is appropriate for data type chosen Note Information is based on data collected at the time the sample was measured and is not modified by a change in cursor position on the Data Viewer real time display Movable x and y axis Available for all data types If the cursor is out of view in either direction rescale the axis by typing over one of the outer limit numbers The cursor absorbance information displayed at the bottom of the page is determined by the position of the movable cursors The movable X determines the baseline from 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 WOR Data Viewer 1 Eia Deefigurstice Daa Papais p irn Dot Boeng S007 EIS rl Peper Mame 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 fo
107. ssneeeesseees 5 1 Mod le et le mec 5 1 Measure F 1 EN 5 1 Blank E28 epus a ES 5 1 ce g pM ea Goledasemeaucee 5 3 Standard e sexies ee ee eee 5 3 NUCICIC AGIOS eM CR Se cence tunseecasmeveuceuensuidens 6 1 Measurement Concentration Range ssssssssessrnrrrreeesrrrrrrreesrrnrrrrene 6 1 Unique Screen Features oeno 6 1 Spectrum Normalization DEE 6 2 Olli ANa E VE 7 1 Fluorescent Dye Selection cccccccccsseseeeceeeeeceeeeeeeeeeeeesaeeeeeeeeeeesaaeaeess 7 1 Measurement Concentration Hange 7 1 Unique Screen Features ained E dE 7 2 Baseline Calculation A Normaltzaton 7 2 VG 8 1 Measurement Concentration Range sssssssssessrnrrrrreesrrrrrrreesrrnrrrreee 8 1 Unique Screen Features ccccccccssssseececeeeeeeeseeeeceeseseeeeeeeeeeeessaaseeeeees 8 1 Protein leede 9 1 Sample Volume Requirements esses 9 1 Measurement Concentration Range ssssssssssessrrrrrrreerrrrrrrreesrrrrrrreene 9 1 Unigue Screen Features ennt octets p ec dean ice abe de pede rad needles 9 2 Spectr m NormaliZatlQri iius eie neca a terere idee ka eate ead inse eier 9 3 lt Proteins EE 10 1 Fluorescent Dye Selection ccccccccseseececeeeeeeeeeeeceeeeseaeeeeeeeeeesesasaaaeess 10 1 Sample Volume Heouiremente 10 1 Measurement Concentration Range sssssssssseessrrrrrreesrrrrrrrrrrenrrrrrrenee 10 1 Unique Screen Feature
108. t check 12V power supply For more details referto the Troubleshooting section of the User s Manual available from Main Menu This error occurs because no light or not enough light is reaching the detector If the troubleshooting steps outlined in the message do not fix the problem perform an Intensity Check e Open the Utilities and Diagnostics module from the main menu e With the sampling arm down select OK to initialize the spectrometer and then select Intensity Check You will see two panels of 8 spectra each and a bias value greater than 65 as shown below This indicates that the USB communication is normal the power supply is operational and the flashlamp is functioning Sun Number Liz 2 SCR Pedes Ho Deech fies 64 Ibn hy Crech H ga uM NES Ail Pade nin IS e a e CR Co vo e D 1 j i i i i 1 1 20 zbo zg ee x50 mbo hn cen se an He Ne Moo xo W lt em Virtanen Accuracy Ar Single Offset DEA Manon Pecks Monitored X P ma ps D pm S ptam GA fel fren 2805 ke miim X21 Bina Hif NS o 1 1 1 1 1 Y f 1 20 300 on Son 45b SOLD SED un un ug 7508 S030 S50 im bh Dad 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
109. t fall within the limits of the standard curve 13 3 Retin to See cle Acuniiton UE Te a EK e Double Chck on any row to change he concentston or delete replicates Ave Abs Abs 1 Abs 2 s jJ Abs Exiting the Bradford Module 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 13 Protein Bradford lt is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module 13 4 Section 14 Cell Cultures 14 Cell Cultures Using an absorbance spectrophotometer to monitor light scattered by non absorbing suspended cells is common practice in life science laboratories Such applications more than any other accentuate the differences between the optical systems of the numerous spectrophotometer designs Note The most distinct difference between the NanoDrop 8000 Spectrophotometer absorbance values for cell microbial cultures and those observed using classical cuvette based systems will be attributable to the shorter pathlength 1 mm vs 1 cm Values may not be exactly 10 fold different as readings are dependent on both the optics of a specific spectrophotometer as well as the cell type in suspension 1 1 IN Di angle 8 rm abs e S angie iD ne ru i 1 A Ei
110. therefore may require special handling to ensure sample homogeneity Effect of Evaporation and Solvents Evaporation of the sample during the measurement cycle usually has just a minimal effect on absorbance readings and may result in a 1 296 increase in sample concentration This can be observed in the field by measuring the same sample successively over time Highly volatile solvents such as hexane will likely evaporate before the measurement can be completed Less volatile solvents such as DMSO can be used successfully 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 advantages of the sample retention system is that samples can be recovered from the upper and lower measurement pedestals by extraction with a pipette Software Architecture and Features Main Menu With the sampling arm in the down position start the operating software by selecting the following path Start gt Programs NanoDrop gt ND 8000 version 3 2 Section 3 General Operation ND 8000 1 0 0 Default Protein User Nucleic Acid A280 Preferences Proteins amp Utilities amp MicroArray Labels Diagnostics Protein Dye Chroma BCA phore Editor Cell Protein Account Cultures Bradford Management Data Protein Viewer Lowry Application Modules The operating software has been tailored to meet the l
111. ting software have the appropriate Windows access level Can t Find LabView RunTime Engine This error message likely means that one or more of the software components have been removed or corrupted If this occurs reinstall the Labview Runtime engine by selecting Start gt Programs NanoDrop Utilities gt Runtime Installer Report Format Loading Error This error occurs when the user does not have Write access to one of the report format files located at c ND 8000 Data custom report formats or the file has been moved from this folder This error is similar to Error Code 8 see above Contact your PC administrator to give all users Read and Write access to this folder Replace the files if they have been removed If the file cannot be located reinstall the software Other Software Error Messages e Source Error This error indicates that there is insufficient light getting through to make good absorbance measurements Check that the sampling arm is in the down position and the power is connected 17 4 e No Active Samples for Measurement This error indicates that there are not any sample positions currently selected for measurement The user may click either All Active or select individual positions for the next measurement 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 operat
112. ulation Beer s Law in the appendix for more details on this calculation e Show Report formatted for 1000 samples although the buffer size can be modified e 260 280 ratio of sample absorbance at 260 nm and 280 nm The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm See 260 280 Ratio in the Troubleshooting section for more details on factors that can affect this ratio Baseline Calculation amp Normalization The software normalizes the visual spectrum display for all readings at 750 nm and will automatically calculate a baseline between 400 and 750 nm for dye concentration calculations 1 2 Section 8 UV VIS 8 UV VIS The UV VIS Absorbance module allows the NanoDrop 8000 Spectrophotometer to function as a conventional spectrophotometer Sample absorbance is displayed on the screen from 220 nm to 750 nm and cursors permit the measurement of individual peaks Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring most aqueous samples However if you are unsure about the surface tension properties of your sample or your p
113. ume 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 recommended that spectrophotometric measurements be made immediately after pipetting samples onto the pedestals as delays can compromise accuracy Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not from properly If this occurs buff the measurement pedestal surfaces by rubbing each measurement surface aggressively with a dry laboratory wipe 30 40 times This will re condition the surface allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found on our website Measurement Concentration Range Using the regular Bradford assay the concentration range of detection is 1
114. uring samples if necessary 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 e Confirm instrument accuracy with CF 1 CF 1 is a concentrated potassium dichromate calibration standard available from Thermo Fisher Scientific and its distributors It is a good practice to check the instrument s performance every six months with a fresh vial of CF 1 260 280 Ratio Many researchers encounter a consistent 260 280 ratio change when switching from a standard cuvette spectrophotometer to the NanoDrop 8000 Spectrophotometer The three main causes for this are listed below 17 7 KKK Change in sample acidity omall changes in solution pH will cause the 260 280 to vary Acidic solutions will under represent the 260 280 ratio by 0 2 0 3 while a basic solution will over represent the ratio by 0 2 0 3 If comparing the NanoDrop 8000 Spectrophotometer to other spectrophotometers it is important to ensure that the pH and ionic strength of an undiluted sample measured on the NanoDrop 800
115. window closed the Sample Position Illuminator will stop flashing 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 12 1 Unique Screen Features file Edt Configuration Standards Help Plank Receding ShmwFHieport User Cirie Deie Timo X31 2007 12 07 FH Exit Fite ID All ducts Cni nmi Gen Make nere DL G Measurement Acie Hj 1 AY emp mi s Go AAG Ha mele Campi ID AGED Hah Hoh Armes L1 H Bl zampe H salam GO AIP Mai mg mL Eagle ID AGED Ha Hah Cannas Ace M 1 cl ampie H D pel ge ONT AA Mai mg ml Saba eee Tanghi AP Wahi Mu m Awe 8 1 Dl Senet n nm late GO Ad Mai i CoS zhandards er J Sengle D Af Hat Hoh Adwe RI JN G zi s ES NEST N 7 NU A E Hak A Sache ID At na i Amwe 4 1 PI Somes
116. with a diamond and cannot be modified Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous nucleic acid samples containing incorporated fluorescent dyes However if you are unsure about the surface tension properties of your sample or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample 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 Sample Detection Limit Approx Upper Weeer Type pmol ul Limit pmol ul SD pmol ul CV Cy5 Cy5 5 and Alexa sample range 0 17 2 4 pmol ul 0 17 Cy3 Cy3 5 Alexa EN Se and Alexa 0 25 100 sample range 0 25 4 pmol ul 0 25 Fluor 660 l sample range gt 4 0 pmol ul 2 5 0 17 60 Fluor 647 sample 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 Alex

NanoDrop 8000 Spectrophotometer V1.1 User`s Manual

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