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NanoDrop ND-1000

1. sessi 43 DU 260 290 ER MO eb oae bcp astutia ren A dun uas oue eat duse Rss 44 643 JUhUsla SDOGIFDIITI sies asus E A EAE taies td natos anco he rusas tuba AE 45 9 TECHNICAL liem 45 7 Maintenance and Warranty ais di didas 47 f A O 47 Vee PARTS THAT REQUIRE REPLACEMENT led 47 5 97 O A uu MIU d MI c Nu M MU 47 T JSECABIBEATION tt al to ees cate eee eee ee 47 1 4 1 LA zero Topgi ro MR 47 V4 2 Paeng ACCUIACY m E 47 8 NanoDrop Technologies Contact Information cesse eeeeeee eren nennen nnn 47 Appendices to NOI GUN a a ten SLE CU 48 As INSTRUMENT SPECIFICA TION Std id 48 B BLANKING AND ABSORBANCE CALCULATIONS cocccccoccncconnnononnnonnnnnonnnnnnnnnnonnnnonnnnnnnnnnnnnnnnnnnnnnnnnenennnnnns 48 C CONCENTRATION CALCULATION BEER S LAW esses nennen nnn 48 D SAMPLE RETENTION SYSTEM SOLVENT COMPATIBILITY ecesssseeee eene nnns 49 E SETTING UP A DYMO 330 LABEL WRITER PRINTER FOR PROPER OPERATION eene 49 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 1 Overview 1 1 Instrument Description The NanoDrop ND 1000 is a full spectrum 220 750nm spectrophotometer that measures 1 ul samples with high accuracy and reproducibility It utilizes a patented sample retention technology that uses surface tension alone to hold the sample in place This eliminates the need for cumbersome cuvettes and ot
2. amp NanoDrop ND 1000 Spectrophotometer V3 0 7 Users Manual NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Table of Contents Mi JOVOIVIOW ena ease a eae III UU III MIN II MINI eecuccd 4 Ted uWNSTSUMENIIDESCISIPTION eee ee ee EIE we EIN T 4 C MEO A TELLE 4 19s wADBBCATIONSE xum ee uM pau DII uM EE M uuu MM 4 NAAN SO i wem e o a S 5 24 SOFTWARE liado ia ide 5 2 1 1 Computer RegulFelmehts s ioca ao A aues a NA 5 Z 12 Hstallallollxi s uccide uM O MON eis I ELE 5 2153 ECONO the Systeri FON auch o Tet ta ener isa pue tub leds uua desc aed 5 214 UDOGIadeS seca penu abii a aida aui Ptr uite ed LadE 6 Ze VI ipm Er 6 2 2 1 6 61 A a ee hea neck Actas thd ea oc 6 2 9 REGISTERING YOUR INSTRUMENT 500 O sees aceasta 6 x A A T Ct 7 3 1 THE SAMPLE RETENTION S YS TEM aceite keiten eee epa E Tutos Ad a 7 out BASIC P 3 1 2 Cleaning the Sample Retention SySteM cooocccconcnocononocononocononononnocononononononanonnanonenanonanass 7 oo salle SiZe REGQUIMCIICINS eyokan E Seer mats edu nts 8 Bai Sample Can VOV TE stance scm mae sb dtoncea ds 8 OO Sample Hotmogellelty io rera n st deed ir qat eiii rete tented eee in uci Med as 8 90 ENECO EVA POAN a 9 Sk Sample Recover aa E 9 3 2 SOFTWARE RCHITECTURE AND FEATURES ata 9 3 2 1 E eet E tensed E E 9 3 2 2 Application MOQOUIOS cas 9 39 ACCOUNTIMANA CEMENTO a 10 3 3 1 SENM UD ACCIONES C 10 CA e
3. Std mg ml 4 000 are measured If erroneous or non 2 ul load up to 5 repli representative readings are encountered for a specific standard all replicates of that ee standard are cleared by selecting Reset this Std Cursors 725 F12 Additionally all m o MEE Standards can be deleted at Md 480 500 520 540 560 Sle 640 660 680 700 720 750 mg ml 4 000 once using the Reset This Window F11 button BCA Protein Zi Step 3 Measure Samples File Edit Show Context Help Once a minimum Standard ge Sese Print Sereen Stat Repor i ve 1 9 2004 3 42 PM Exit Measure i 1 a Standard C andard Curve User Default curve has been established J Sampe the red indicator light will turn Sample m i green allowing the user to m either start measuring Batch Index 0 samples Sample concentrations are calculated using linear interpolation point to point between the Dust aaro vt poly rra two Standards flanking the unknown Sample Note In order to obtain a Absorbance NN concentration value mg ml i the sample unknown must Cursor 4 725 fall within the limits of the Absorbance 0 002 Standard curve 0 02 p l l l 1 I l l 1 l 1 mu 450 480 500 S20 540 560 580 600 620 640 660 680 700 720 750 mg ml 0 318 3 0 0 B1289 Wavelength nm s 4 6 7 Standard Curve Features Unlike the Bradford Protein assay the BCA Protein Assay 562 nm is much more linear throughout
4. 4 0067 8 0 04 i i 1 r 1 i i 1 t i 1 i 0 500 1000 1500 2 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 ug ml Absorbance ug ml AveAbs Abs 1 Abs2 Abs 3 Abs User Default Reference 0 ung aor jooo joo Standard 1 2 0 079 0080 10 078 Stendwd2 150 0112 0113 J012 atte ll Standard 3 100 0128 0128 0123 3 0 0 A152 eden I 34 Standard 5 ua iiis 11 5 2003 1 49 A mini Bradford assay covers an approximate range of 15 100 ug ml e e o y ol a odi Eee Ee o w o 4 7 8 Exiting the Bradford Module Be sure not to Exit the Bradford software module until you ve processed all the unknowns to be assayed The Standard Curve is automatically deleted when the software is closed and cannot be recalled 4 8 Cell Cultures Module 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 differences amongst the optical systems of the numerous spectrophotometer designs The Cell Cultures module displays the sample spectrum from 250 nm to 700 nm One cursor is fixed at the frequently used wavelength for monitoring cell suspensions 600nm while the second cursor can be set to the wavelength of interest 4 8 1 Sample Size Requirements Field experience h
5. Microsoft Word For Windows check box at bottom called Always use Copy Proof Windows Meda layer this program to open these files Select Create Shortcut e a v A thi t t i OK From now on double clicking a ndt P pube MM MEME file will open the file in Excel Ic Cancel other 3 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 6 Troubleshooting 6 1 Signal Error See Signal Error This is most likely caused by Sample surface is dirty make sure surfaces are clean Communication error quit ManoDrop software unplug the USB cable wait 20 sec connect USB cable and restart software Wo power to instrument check 12 power supply Sample arm make sure sample arm is in down position Refer to Ehe Troubleshooting section of Ehe users manual For more detail Refer to wew nanodrop com Far manual rev 16 Apr 04 This is the most common software error encountered and usually occurs when starting It occurs because no light or not enough light is reaching the spectrometer or because the USB communication has not been established or has been lost Try the following troubleshooting steps to diagnose and fix the problem 1 Confirm that the sampling arm is in the down position 2 vigorously with de ionized water and a laboratory wipe 3 Clean the Measurement Pedestal Surfaces A sample may have dried on one or both of the sample pedestals and is now absorbing the light Clean
6. O Abs 2 0 002 wus y A a 1 i TE i A 3 0 0 81194 Wavelength nm 21 Abs1 and A2 Abs2 current values of the user selectable wavelength cursors and corresponding absorbances The wavelengths can be set by dragging the cursor using the up down arrows or typing in the desired wavelength Baseline the absorbance of the user selectable baseline horizontal cursor The user may drag this cursor to a new vertical position to create a new baseline The absorbance value of the baseline is subtracted from the absorbance of the spectrum Max Absorbance used to rescale the upper limit of the vertical axis Hi Abs samples with high absorbance up to 75 A equivalent 10 mm path can be measured directly applicable for instruments with serial number gt 500 only or that have been field retrofitted This capability is selected by choosing the Hi Spec button on the header bar When this is selected the absorbance is measured using the short path 0 2mm and plotted as a red line as shown above Sample ID label will be stored with sample data in the file folder C NanoDrop Data Username UV Vis HiAbs Normalize this is the only module where this is a user selectable feature If selected the software will automatically normalize the spectrum based on the lowest absorbance value in the range 400 700 nm 4 5 Protein A280 Module Proteins unlike nucleic acids can exhibit considerable diversity The A280 method is
7. tab Ensure that Enable advanced printing features box is checked Then click on Printing Defaults then Advanced Then set the following e Paper Size 30256 e Halftoning Super Cell e Print Quality Barcodes and Graphics Click OK to close this window then click Apply Last click on the Device Settings tab and ensure 30256 Shipping Label is selected as the Default label rev 16 Apr 04 50
8. the assay s range and has approximately 10 fold more signal available A representative standard curve is shown The Standard Curve can be printed but not saved for later use since its utility is limited to the specific time period in which it and the respective unknowns are assayed 28 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 BCA Protein Std Curve x Absorbance mg ml AveAbs Abs Abs2 Abs 3 Abs 4 Abs User Default Reference 0000 003 0013 003 Standard 1 0200 0022 0022 0022 0023 Standard 2 1000 0163 0163 0163 0163 11 5 2003 3 04 Standard 3 2000 0317 0313 0320 10320 SANAE e e e m m mend lt 1 00 020 caine Regular BCA Standard Curve 0 2 i a a IA CEET ee l 8 0 mg ml 0 70 0 60 0 50 0 40 0 30 1 mm Absorbance 0 20 0 10 gt A 0 00 0 10 cl i i 0 00 0 50 1 00 BCA Protein Std Curve Absorbance mg ml AveAbs Abs 1 Abs 2 Abs 3 User Default Reference 0000 0 002 0002 0003 0003 Standard 1 0025 0068 0068 0 069 0 069 Standard 2 0050 0138 0135 loisi 0136 0133 0137 Standard 3 0100 0242 0241 0241 0245 3 0 0 C004 O IMEULT 0420 0423 0423 ew 0 45 0 40 i a ae E a TT f T Tode mini BCA Standard Curve 0 01 m g Fe T 0 20 mg ml e E 0 30 G 11 7 2003 11 48
9. this will account for 1 2 increase in sample concentration This can be observed in the field by measuring the same sample successively over time 3 1 7 Sample Recovery One of the advantages of the sample retention system is that the undiluted samples can actually be recovered from the upper and lower measurement pedestals by extracting with a pipette 3 2 Software Architecture and Features 3 2 1 Main Menu With the sampling arm in the down position start the NanoDrop software by selecting the following path Start gt Programs gt NanoDrop gt NanoDrop 3 0 1 2101 File Help User Default administrator Nucleic Acid Protein User Measurement AZUL Preferences Micron Array Protein Account Measurement BLA Management Uv vis Protein Utilities amp Measurement Bradford Diagnostics Change Password Cell Cultures 3 2 2 Application Modules This software has been tailored to meet the life scientist s needs It includes the following application modules e Nucleic Acid Measurement concentration and purity of nucleic acid MicroArray Measurement dye incorporation concentration and purity of nucleic acid General UV Vis Measurement general UV Vis measurements Protein A280 concentration and purity of purified protein Protein BCA protein concentration using the BCA assay Protein Bradford protein concentration using the Bradford assay Cell Culture absorbance measurement of suspended cells Nan
10. 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 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 represented as if measured with a 10 mm path Note this is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length 260 280 ratio of sample absorbance at 260 and 280 nm The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm See 260 280 Ratio section of the Troubleshooting section for more details on factors that can affect this ratio 260 230 ratio of sample absorbance at 260 and 230 nm This is a secondary measure of nucleic acid purity The 260 230 values for pure nucleic acid are often higher than the respective 260 280 values They are commonly in the range of 1 8 2 2 If the ratio is appreciably lower this may indicate the presence of co purified contaminants ng ul sample concentration in ng ul based
11. Occurred Code 7 Source Open file in This error occurs when any of the five log files dye list log passwords log user preferences log protein methods log and photo methods log has been removed from the folder where the program files were installed default is Program Files NanoDrop 3 0 1 These files must be present in this folder in order for the NanoDrop software to operate 6 2 4 An Error Occurred Code 8 Source Open file in This error occurs when access to any of the five log files dye list log passwords log user preferences log protein methods log and photo methods log has been denied This usually occurs when a user without administrator access runs the software If this is the case contact your system administrator to share the program files were installed default is Program Files NanoDrop 3 0 1 This error can also occur if the log files are set to read only Check this by going to the NanoDrop 3 0 1 folder and right click on the dye list log file Deselect the read only box if it was selected Repeat this for all of the log files 6 2 5 Source Error This error indicates that there is insufficient light getting through to make good absorbance measurements or the USB connection has been lost Check that sampling arm is in the down position and the power is connected If the USB connection has been lost the Low Detector Bias error will also app
12. Plugged in E Running on Options Change the I batteries System Standby and Turn off monitor After 20 mins After 15 mins oystem Hibernate to Turn off hard disks Never y After 10 mins never for the Plugged In System standby Never y After 30 mins column as shown at right System hibernates Never y After 45 mins L Faulty USB card on PC Confirm that the USB is faulty by trying to operate the instrument on a different PC that is known to have properly functioning USB ports If necessary replace the USB card on the PC 6 4 Data Concerns 6 4 1 Sample Accuracy and Reproducibility Most of the occurrences of strange data are a result of sample preparation All of the causes listed below can potentially cause apparent problems with data Possible Cause Explanation and Possible Solution Sample is not homogeneous Due to the small volumes required by the ND 1000 it is extremely important to ensure that the sample being measured is homogeneous Field experience has shown that samples containing large molecules such as genomic or lambda DNA are particularly susceptible to this phenomenon Note that the larger volumes used by cuvette based spectrophotometers will minimize or mask the effect of sample non homogeneity Liquid sample column breakage Very strange results can occur when the liquid sample column is not completely formed during a measurement While making a measurement visually confirm that t
13. 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 program Save this as a jpg or doc file and send as email attachment to your distributor or to info nanodrop com 3 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 distributor or to info nanodrop com 46 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 7 Maintenance and Warranty 7 1 Cleaning The primary maintenance requirement of the NanoDrop ND 1000 Spectrophotometer is keeping the measurement pedestal surfaces clean Upon completion of each sample measurement wipe the sample from the upper and lower pedestals to prevent sample carryover and avoid residue buildup It is also recommended that both measurement pedestals be cleaned with de ionized water upon completion of a testing series No other regular maintenance is required 7 2 7 3 Parts That Require Replacement In general the only part that should periodically require replacement is the flash lamp The flash lamp should last a minimum of 30K measurements There is no way to test the flash lamp to confirm its remaining life When the flash lamp fails the light output will become very erratic or stop alto
14. USB2000 3 0 0 0 0001 ESA 11107200 4 27 PM 0 300 0 001 700 0 001 Measure USB2E31 USB2000 3 0 0 0 0001 4 11 10 200 4 34 PM 0 454 0 004 700 0 003 Blank USB2E31 USB2000 3 0 0 0 0001 05 5xdil Fluorescein 11 10 200 4 36 PM 0 490 1 501 700 0 002 Measure USB2E31 USB2000 3 0 0 0 0001 6 5xdil Fluorescein 11 10 200 4 36 PM O 490 1 501 700 0 003 Measure USB2E31 USB2000 3 0 0 0 0001 Z 5xdil Fluorescein 11 10 200 4 37 PM 0 490 1 515 700 0 003 Measure USB2E31 USB2000 3 0 0 0 0001 8 5xdil Fluorescein 11 10 200 4 38 PM 490 1 52 700 0 003 Reblank USB2E31 USB2000 3 0 0 0 0001 5 1 4 Automatically Opening Archive Files with MS Excel To set up your PC to automatically open archive files with MS Excel the ndt extension must first be associated with MS Excel To do this perform the following E3 Microsoft Excel For Windows GH Microsoft FrontPage JE Microsoft Image Composer Open with 0554 1 Go to the C NanoDrop Data Default en i f i i New Click the program you want to use to open directory and find any ndt file Right Print A Nucleic Acid 2003 09 09 ndt cl ick on the fi le to b ri n g u p th e menu a t Dsen dita Fay Recien If the program is not in the list click Other i dl i ij Open With Choose the program you want to use right Select Open with TL Scan with Norton AntiVirus 2 Choose MS Excel or other Send To Wf Microsoft Paint E spreadsheet prog ram and select the Cut Gi microsoft PowerPoint For Windows fF
15. applicable to purified proteins exhibiting absorbance at 280nm It requires no prior calibration 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 is the only other NanoDrop software module that displays and records 10 mm 1 cm equivalent data on the screen and in the archived data file 4 5 1 Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension That occurrence can be overcome without 23 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurement 4 5 2 Special Cleaning Requirements for Proteins Proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form If this occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 2
16. are normalized to a 1 0 cm 10 0 mm path For MicroArray UV Vis BCA Protein Bradford and Cell Culture Application modules the data is normalized to a 0 1 cm 1 0 mm path For high absorbance UV Vis samples data are normalized to a 0 1 mm path Note 2 for the UV Vis and Hi Abs archive data a column entitled Measure Type is displayed 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 For example in the image below row 8 contains the absorbances for the Reblanked sample 5x dil Fluorescein utilizing the new blank while row 7 is the initial absorbances of the same sample utilizing the original blank value 36 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 24 Microsoft Excel U is 2003 11 10 ndt EE 13914 6597 File Edit View Insert Format Tools Data Window Help Acrobat l X DHS SRAY X86 o amp r 5 M c IE A29 pee UE Se Se SS SS J K L MT 1 Sample ID Date Time Baseline Cursor 1 Pos Cursor 1 Abs Cursor 2 Pos Cursor 2 Abs Measure Type Serial Firmware Software Confic E 11 10 200 4 26 PM 0 300 0 002 700 0 005 Blank USB2E31
17. both of the sample pedestals gently but Confirming Resetting the USB Communication With the sampling arm down open the Utilities and Diagnostics Module Select OK to initialize the spectrometer and then select Intensity Check The USB communication is functioning properly if screen is similar to the one below left The bias value should be greater than 65 counts Detector Bias 4000 0 LE Jl E NW e m S00 0 M x 1000 0 0 0 A LU i I I LU LU L 1 j L 230 0 300 0 350 0 400 0 450 0 500 0 550 0 600 0 650 0 700 0 750 0 Wavelength nm Serial Number Configuration USB2G1212 B1154 1 002097 0 31 64 24 Proper Operation The detector bias is low contact vour distributor or the NanoDrop Technologies Inc about service No USB Communication If the Bias is less than 65 counts or you receive a Low Bias error above right this indicates no USB communication between the PC and instrument Try the following possible remedies to correct the problem e Confirm that the USB cable is connected to both the PC and instrument 38 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 e Close the NanoDrop software unplug the USB cable wait 20 seconds reconnect the USB cable and restart the software e Restart the PC e Reset the spectrometer configuration see step 4 below e If steps above do not correct the problem uninstall the NanoDrop software and the OOlBase32 software and restart
18. delivering 1 microliter to the measurement pedestal If you are unsure about your sample characteristics or pipettor accuracy a 2 ul sample is recommended 3 1 4 Sample Carryover Prevention of sample being retained on the ND 1000 Spectrophotometer s measurement pedestals is easily addressed Simple wiping of the upper and lower measurement pedestal with a dry laboratory wipe is highly effective in eliminating carryover for samples differing in concentration by as much as three orders of magnitude see data at www nanodrop com This is possible since each measurement pedestal is in actuality a highly polished end of a fiber optic cable There are no cracks or crevices for residual sample to reside in 3 1 5 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 acid such as resuspended nucleic acid preparations are common examples known to the molecular biologist Proteins are subject to denaturation precipitation and aggregation so they too have special handling requirements to ensure sample homogeneity NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 3 1 6 Effect of Evaporation Evaporation of the sample during measurement has a very small effect on results Typically
19. on absorbance at 260 nm and the selected analysis constant 19 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 2 4 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 4 3 MicroArray Measurement Module The capability to pre select viable fluorescent tagged hybridization probes for gene expression in microarrays can eliminate potentially flawed samples and improve research effectiveness The NanoDrop software facilitates the measurement of DNA concentration and dye labeling effectiveness The NanoDrop ND 1000 Spectrophotometer measures the absorbance of the fluorescent dye allowing detection at dye concentration as low as 0 2 picomole per microliter 4 3 1 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 dye However if you are unsure about your sample or your pipettor accuracy a 1 5 2ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample 4 3 2 Measurement Concentration Range The NanoDrop ND 1000 Spectrophotometer will accurately measure fluorescent dye and nucleic acid concentrations up to 100 pmols ul Cy3 and 750 ng ul DNA re
20. ozs 0 20 t 7 0 10 0 05 0 00 i 1 i i i i i i i I i i 1 I i 0 00 0 01 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 09 0 10 0 11 0 12 0 13 0 14 0 15 0 16 0 17 0 18 0 19 0 20 mg ml 4 6 8 Exiting the BCA Module Be sure not to Exit the BCA software module until you ve processed all the unknowns to be assayed The Standard Curve is automatically deleted when the software is closed and cannot be recalled 4 7 Protein Bradford Module The Bradford Assay is a second alternative method commonly utilized for determining protein concentration It is often used for more dilute protein solutions where lower detection sensitivity is needed and or in the presence of components that also have significant UV 280 nm absorbance Like the BCA method the Bradford method requires Standard Curve generation each time the assay is run before unknown proteins can be measured The Bradford uses the protein induced Absorbance shift of Coomassie Blue dye to 595 nm as a measure of protein concentration 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 amp Samples unknowns ensuring they are subjected to the identical conditions and timing throughout the assay 4 7 1 Sample Volume Requirement Some proteins are hydrophobic and others hy
21. to the User name on the main menu User Default 3 3 5 Account Lockout User specific accounts can become locked out in several ways for example e failure to change password within the allotted time e incorrectly entering the password 99 consecutive times e the Administrator locks a specific account Only the Administrator level 10 can unlock a locked account This is done by using the Modify User entry in the Account Management module Note all accounts even the Administrator can be locked if the incorrect password entry described 3 3 6 Password log file This file contains the respective User ID amp password for all accounts It is located within the Programs Folder under the NanoDrop 3 0 1 folder Itis strongly recommended that the Administrator make a copy of that file and store it in the same folder each time a New User account is added If the Administrator s account becomes locked the up to date copy of the password log file can be renamed and used as the password log file Otherwise a blank password log file can be obtained from the NanoDrop software CD or downloaded from www nanodrop com In this case all user accounts would need to be rebuilt by the administrator Note If upgrading to V3 0 0 from V3 0 1 the passwords log and user preferences log files should be automatically copied to the proper directory for use with V3 0 1 If this occurs properly then all the user accounts that were created w
22. 0 0 B1312 Wavelength nm 600nm Absorbance current value of the absorbance at the 11 cursor with the Baseline absorbance subtracted Note the actual 1 mm absorbance is displayed and Absorb current value of the user selectable wavelength cursor and corresponding absorbance The wavelength can be set by dragging the cursor using the up down arrows or typing in the desired wavelength Note the user selected wavelength and absorbance are not utilized in any calculations Baseline the absorbance of the user selectable baseline horizontal cursor The user may drag this cursor to a new vertical position to create a new baseline The absorbance value of the baseline is subtracted from the absorbance of the spectrum Max Absorbance used to rescale the upper limit of the vertical axis 4 8 4 Sample Homogeneity The user must be sure to homogeneously suspend the cells when sampling for absorbance measurement and read the sample immediately to avoid significant cell settling Vigorous mixing may be required particularly when measuring concentrates 34 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 5 Archived Data Sample data from all Application modules is automatically stored in archive files The data in these files can then be manipulated by MS Excel or other spreadsheet program 5 1 1 Data Storage Hierarchy The hierarchy for archive files is as follows C NanoDrop Data gt Username gt Appli
23. 0 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 6 1 Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension That occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2ul sample size is recommended for protein measurement 4 6 2 Special Cleaning Requirements for Proteins Proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form If this occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 20 times This will re condition the surface allowing for the liquid sample column to form 4 6 3 Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer the regular BCA assay can run from 0 20 mg ml up to 8 0 mg ml A mini BCA assay covers an approximate range of 0 01 0 20 mg ml Approximate Approximate Typical Reproducibility Assay Type Lower Limit Upper Limit minimum 5 replicates mk a ECCO ee mg ml CV Regular BCA Regular BCA 29e over entire 296 over entire range Mini BCA e 0 01 mg ml over entire range 4 6 4 BCA Kits Protocols and Sample Preparation Commercia
24. 0 times This will re condition the surface allowing for the liquid sample column to form 4 5 3 Measurement Concentration Range The NanoDrop ND 1000 Spectrophotometer will accurately measure protein samples up to 100 mg ml BSA without dilution To do this the instrument automatically detects the high concentration and utilizes the 0 2mm pathlength to calculate the absorbance A table of concentration range and typical reproducibility is listed below Detection Approximate ny peal proper Sample Type diis t minimum 5 replicates Limit Upper Limit SD mg ml CV 9 4 a sample range 0 05 10 mg ml 0 10 mg ml Purified BSA 0 10 mg ml 100 mg ml sample range 10mg ml 2 i Mak Suwnepon Mas nane 6 02 AM wt Report Show Report Measurement User Default SampleType Abs 1 mg mL BSA IgG Lysozyme Other protein E 1 Other protein E amp MW A 280 Abs 0 000 A 280 10 mm path NaN 260 280 NaN 0 107 1 1 1 1 i 1 1 4 220 230 240 250 260 270 280 290 300 310 320 330 340 350 mg ml 32Beta A152 Wavelength nm 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 select
25. 2 establishes a new reference blank that is used for the absorbance calculations of subsequent samples However unlike the Blank F3 function the Re blank feature recalculates the absorbance spectrum for the most recent sample and displays this on the screen When the Re blank function is used the following message appears Blank Applied To displayed Spectrum See the Blanking and Absorbance Calculations appendix for more information on absorbance calculations 4 1 6 Print Screen F4 The Print Screen button will print a copy of the current operating screen to the default printer attached to the operating PC Note The system is configured to work with the Dymo LabelWriter 330 printing on 30256 2 5 16 X 4 shipping labels but can print on any printer connected to the PC 16 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 1 7 Start Report F6 The user can log up to 32 measurement results and print them to the desired printer To initiate this feature select the Start Report button Once selected this button will read Recording and will stay in this state until deselected Once selected a Sample Report will automatically print to the Default printer when the maximum number of entries for that specific report has been reached The Batch Index number will automatically reset the entries in the Sample Report will be cleared with the next sample measured
26. A A an tie cta su eU Td EM 11 E ACCOUN oor ect 11 9249 4 JJACCOUDL LOTOU rro ae Aue 12 Fao ACCOUN LOCK OU puc P QN 12 919 0 fasswordlog lg ee A i tiet editt e Ee e iE etes uus 12 OU WISER PREREREN CES iS E M cdd nere Dah eh E od med AE 12 39 CHANGE PASSWORD i550 a cvtiatiee tavi eet aioe et len Sed oi ee aes 13 9 0 WTIEMIESAND DIAGNOSTICS saos Senet di 14 ee AS ee AA RT eee ee 15 4 Applications Modules cuina id ideo a 15 4 1 FUNCTIONS COMMON TO EACH APPLICATION MODULE occcccoccnncccnnncconnncnnnnnonnnnnonnnnonnnnonononononcnnnos 15 4 1 1 Module Sta UD A A A A en nea 15 442 Escape Koy ESC A a 15 ATI MOISES 15 AA Blank F3 sinees es 15 4 1 5 Reblank F2 riiin PP 16 4 1 6 AS EEN Ad 16 ASE Slan ROO 17 41 8 Print Report F9 ii iaa 17 Bets JSHOWTSODOFIL S T issue vd uc MM ee 17 Jd MI Sample JD Hane M ium MM M MEE 17 Z dd SBOICIHIDOOX xiii MICE UM M IM 17 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 41 12 Printing Vid TIC Pint I CU caves ean ete A avai ait Met benidi edu aged d undi 18 AMAS ME A A Rd 18 A AD Conex O Dt cias 18 4 2 NUCLEIC ACID MEASUREMENT MODULE cccooccnccccnnccnonnnconnncnonnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennninenos 18 4 2 1 Sample Volume Requirements cccoonncccocnnococonoocnnocononononononanononcnnonononononononanononanno
27. Ext Coeff E 1 Liq m cm 10 00 protein and A current value of the user selectable wavelength cursor and corresponding absorbance The wavelength can be set by dragging the cursor using the up down arrows or typing in the desired wavelength Note the user selected wavelength and absorbance are not utilized in any calculations A280 10 mm Path 10 mm equivalent absorbance at 280 nm for the protein sample measured A260 280 ratio of sample absorbance at 260 and 280 nm 4 5 5 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 4 6 Protein BCA Module 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 Standard Curve generation each time it is run before unknown proteins can be measured The resulting Cu BCA chelate formed in the presence of protein is measured at its wavelength maximum of 562 nm Pre formulated reagents of BCA and CuSO utilized in the assay are available in kit form from numerous manufacturers Follow their recommendations when mixing the respective reagents at the time the assay is to be performed 25 NanoDrop ND 100
28. 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 the 260 280 ratio For example a 1 nm shift in wavelength accuracy will result in a 0 2 change in the 260 280 ratio Since many spectrophotometers claim a 1 nm accuracy specification it is possible to see as much as a 0 4 difference in the 260 280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are both within wavelength accuracy specification Nucleotide mix in your sample The five nucleotides that comprise DNA and RNA exhibit widely varying 260 280 ratios The following represent the 260 280 ratios estimated for each nucleotide if measured independently Guanine 1 15 Adenine 4 50 Cytosine 1 51 Uracil 4 00 Thymine 1 47 The resultant 260 280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260 280 ratios for the four nucleotides present It is important to note that the generally accepted ratios of 1 8 and 2 0 for DNA and RNA are rules of thumb The actual ratio will depend on the composition of the nucleic acid Note RNA will typically have a higher 260 280 ratio due to the higher ratio of Ura
29. able to find device with serial oooccccocccccocnnoconononononononnnnanonnns 40 62 2 Cantina tile OOTDENWV INI icto etate te etd do tee eren A iaa 40 6 2 3 An Error Occurred Code 7 Source Open file D ooccoconcccconnnooccnocononecononocononononns 40 6 2 4 An Error Occurred Code 8 Source Open file D ooccococccoconnnoocnnocononecononocononononns 40 029 SOUCO ENON oct eet ae eee o ULL MCA EE 40 02 06 A 41 6 2 7 Error 7 Occurred at New File u a a a a a a A E E 41 020 ISR INOCINS AICO a atte a ee E E abt abus EE wat eee 41 6 2 9 Can t Find LabView RUNTIME ENgiN occccoconcncconcccconincoonononononononononononononononononannnnaninnnons 41 6 2 10 EZUSB SYS Cannot Be Fold eor er Pe E aaa 41 6 2 11 Driver X Configuration Failed You Must Manually Edit the Registry 42 6 2 12 Error 8 Occurred at Open File siaii a a a iaaa aaa ossa 42 6213 JBDSUflcient MORIOFV assistenti eie A EA AAA EAN DDR AAA AAA 42 0 2 14 ENI COIE S T etit tet uon iie toD ohm tef aula a 42 D A DNAT E cet uuu sti cpm mutus Mu LE M DIS ee Le 42 6 3 1 Ghaltenhg SOIGhold sess tet uo on binds tees tank maie ueab ette tutis eo haeres unu asa dedu Re 42 6 3 2 Intermittent USB Connection Failure occccconcccccnnocononocononecononoonononononononononanononannonanoss 42 D UDADAXONGERNSC estat its deat ad eut cuf pneu M pte 43 6 4 1 Sample Accuracy and Reproducibility
30. and the respective parameters will begin to populate the next Sample Report 4 1 8 Print Report F5 Selecting the Print Report F5 button will print the existing Sample Report on the default printer if there are any samples in the Sample Report It does not clear the Sample Report contents Note that the batch logging holds a maximum of 32 samples A Sample Report will automatically print to the Default printer when the maximum number of entries for that specific report has been reached If the PC is not connected to a printer you will see the following error message RS a An error occured code 34 Source Invoke Mode in Print Panel vi z VI GUI Printer vi gt U Vis Report vi zLIy Vis wi Manabrop 3 0 41 This is a non fatal error and will not cause the software to shut down Note The system is configured to work with the Dymo LabelWriter 330 printing on 30256 2 5 16 X 4 shipping labels but can print on any printer connected to the PC 4 1 9 Show Report F7 At any time the user can display the entries comprising the current Sample Report by selecting the Show Report button Descriptive parameters specific for the individual Application modules are populated for each individual Sample ID There are three options within the Show Report window e Save allows the user to save the existing report e Print prints current report to default printer e Exit
31. as indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous samples However if you are unsure about your sample composition or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path completely covered by sample 33 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 8 2 Cell suspension Concentrations Due to its shorter pathlength the ND 1000 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 280 nm 4 8 3 Unique Screen Features Cell Cultures Module T X File Edit Show Context Help p er Measurement complete 179 2004 3 02 PM Exit Measure User Default J 0 70 Max Absorbance Sample ID 0 70 CC 05 6 Bil 3A A Wai aa a Batch Index 2 0 50 Baseline 0 000 0 45 AT Bm is 0 034 0 35 E aa Aass Absorb E 025 2 0207 an User Cursor 015 s BM A 3280 nm BB gt Mac Absorb 0 468 4 D 07 LU I 1 1 I l Lu 1 LU LU LU 1 LU 1 1 LU LU 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 3
32. asurement 0 2mm Sample Size Requirement 1 2 ul Light Source pulsed Xenon flash lamp Detector 2048 element linear silicon CCD array Wavelength Range 220 750 nm Wavelength Accuracy 1 nm Wavelength Resolution 3 nm FWHM at Hg 546 m Absorbance Precision 0 003 absorbance Absorbance Accuracy 2 at 0 76 absorbance at 257 nm Absorbance Range 0 02 75 10mm equivalent absorbance Measurement Cycle Time 10 seconds Dimensions 20 cm X 15 cm x 12 cm Sample Pedestal Material of Construction 303 Stainless Steel Fiber Optic Cable Material of Construction quartz Operating Voltage 12 Vdc Operating Power Consumption 6W Standby Power Consumption 1 5W 8 2 B Blanking and Absorbance Calculations When the NanoDrop ND 1000 Spectrophotometer is blanked a spectrum is taken of a reference material blank and stored in memory as an array of light intensities by wavelength When a measurement of a sample is taken the intensity of light that has transmitted through the sample is recorded The sample intensities along with the blank intensities are used to calculate the sample absorbance according to the following equation Absorbance log Intensitysampie Intensitypiank Thus the measured light intensity of both the sample and of the blank are required to calculate the absorbance at a given wavelength 8 3 C Concentration Calculation Beer s Law General The Beer Lambert equation is used to correlate the calculated absor
33. ath 0 007 ZE 260 230 0 29 0 10 lo 2h alo zh ao o dh lo la alo xb ob oo oo MAL A 3 0 0 C001 Wavelength nm Initial blank should produce a straight line Subsequent blank should produce a nearly straight curve that is very close to baseline Blanking Cycle For the most consistent results it is best to begin any measurement session with a blanking cycle This will assure the user that the instrument is working well and that any sample carryover from previous measurements is not a concern To perform a blanking cycle perform the following 1 Load a blank sample the buffer solvent or carrier liquid used with your samples onto the lower measurement pedestal and lower the sampling arm into the down position 2 Click on the Blank F3 button in the Application module you have open 3 Analyze a fresh replicate of the blanking solution as though it were a sample This is done using the Measure button F1 The result should be a spectrum that varies no more than 0 050 A Nucleic Acids and Protein A280 10mm path equivalent or 0 005 A all other Application modules 1mm path from the zero baseline 4 Wipe the blank from both measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum is within 0 005 A 1mm path See the Blanking and Absorbance Calculations appendix for more information on absorbance calculations 4 1 5 Re blank F2 The Re blanking option F
34. bance with concentration A E b c where A is the absorbance represented in absorbance units A E is the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of liter mol cm b is the path length in cm and c is the analyte concentration in moles liter or molarity M Fluorescent Dyes Microarray Measurement The NanoDrop software uses the general form of the Beer Lambert equation to calculate fluorescent dye concentrations in the Microarray Concentration module The table of extinction coefficients for each dye is below 48 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 ye lye liter mol cm Wavelength nm Nucleic Acids For nucleic acid quantification the Beer Lambert equation is manipulated to give c A e b Where c is the nucleic acid concentration in ng microliter A is the absorbance in AU e is the wavelength dependent extinction coefficient in ng cm microliter and b is the path length in cm The generally accepted extinction coefficients for nucleic acids are e Double stranded DNA 50 e Single stranded DNA 33 e RNA 40 For the NanoDrop ND 1000 Spectrophotometer a path of 1 0 mm and 0 2 mm are used compared to a standard spectrophotometer using a 10 0 mm path Thus the NanoDrop ND 1000 Spectrophotometer is capable of measuring samples that are 50 times more concentrated than can be measured in a standard spectrophotometer Note absorbance
35. ble measurement range Broken fiber optic cable If the upper black fiber optic cable is broken you can get widely varying results If you suspect that this is occurring refer to the Technical Service section for instructions on how to contact NanoDrop Technologies and what information must be sent to allow for diagnosing the problem If the fiber optic cable is broken it must be replaced USB connection drops out Close NanoDrop software unplug USB cable wait 20 seconds causing solenoid failure during reconnect cable wait 1 minute and restart software With measurement Windows 98 amp ME PCs you will need to restart the PC 6 4 2 260 280 Ratio Many researchers encounter a consistent 260 280 ratio change when switching from a standard cuvette spectrophotometer to the NanoDrop ND 1000 spectrophotometer The three main causes for this are listed below Change in sample acidity Small changes in solution pH will cause the 260 280 to vary An acidic solution will under represent the 260 280 ratio by 0 2 0 3 while a basic solution will over represent the ratio by 0 2 0 3 If comparing the NanoDrop ND 1000 Spectrophotometer to other spectrophotometers it is important to ensure that the pH of an undiluted sample measured on the NanoDrop ND 1000 Spectrophotometer is at the same pH as the diluted sample measured on the second spectrophotometer William W Wilfinger Karol Mackey and Piotr Chomczynski Effect of pH and lonic
36. cation Module BCA Protein Bradford Cell Culture MicroArray Nucleic Acid Protein A 280 UV Vis or UV Vis HiAbs All archived data files are stored within an Application Module folder that is within the User folder as shown below Back p O Search CE Folders Ea ie ul xn EE Address Default e an Norton AntiVirus Folders 50 EH Download BCA Protein File Folder 10 3 2003 4 09 PM BM DRIVERS Bradford File Falder 8 20 2003 9 05 AM BERT o Cell Culture File Falder 8 20 2003 9 33 AM A INET MicroArray File Falder 3 17 2003 3 34 PM BM Multimedia Files Nucleic Acid File Folder 9 9 2003 5 19 PM EB NanoDrop Data Protein A 280 File Folder 8 20 2003 9 34 AM o0 Sap acivinicteatar LIN Vis File Falder 8 20 2003 9 33 AM Bug LIN Vis HiAbs File Falder 10 14 2003 1 17 PM EHE Jeff amp 7 Leslie EHC Philippe Fl Palm 5 1 2 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 2003 12 03 ndt corresponds to Nucleic Acid data from the software session that began on December 3 2003 A unique file extension ndt has been given to these files to enabl
37. cil compared to that of Thymine i Leninger A L Biochemistry Qn ed Worth Publishers New York 1975 44 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 6 4 3 Unusual Spectrum A sample that exhibits jagged cuts out of the spectrum but an otherwise normal shape may be the result of detector saturation This can be caused by the software selecting too high of an integration time due to a dirty sample pedestal upon startup Try cleaning both sample pedestals thoroughly and restarting the software For reference examples of spectra generated with a saturated detector are shown below r Nucleic Acids 12 16 2003 3 39 PM 3 39 PM Lex Print Fogar 1279 2003 4 45PM est int Rep Repon Standard Curve 7 User Default User Default 2 0 50 Max Absorbance Y Ready for Sample J Standard 5 0 50 Replicate I Std ug ml 2000 sing L load up to 5 replicates DI s and measure A230 Abs 1592 A260 10 mm path 3111 426010 mm path 1946 260 280 1 60 260 230 1 94 e z lis 1 y D t i D 1 ng uL 1555 220 230 240 250 260 270 260 290 300 310 320 330 340 350 3 0 0 C001 Wavelength nm Detector saturation nucleic acid measurement Detector saturation Bradford measurement A spectrum that is very un smooth or ragged can be caused by insufficient light intensity reaching the spectrometer If you suspect that this is occurri
38. cturers guidelines a Standard Curve is required every time the Bradford Assay is run Single or Multi point calibration is incorporated into the software An operable Standard Curve can be developed using a single replicate of the Reference Bradford reagent only no protein and a single replicate of one Standard The multi point calibration allows a maximum of five 5 replicates for each of five 5 different Standards There is no set order in which Standards must be run Using NanoDrop version 3 0 1 software there are only three 3 general procedural steps to unknown protein concentration measurement The requisite order including generating the standard curve is as follows Protein Bradford o xi UTEDTTIUUTUNUTPNT Step 1 Measure the Reference Bradford p 179 2004 3 02 PM Exit reagent a zero Measure Standard Curve User Default Standar d Max Absorbance Q Apply more standards J VINE l The software will not allow l measurement of Samples i until at least 1 Standard and Reference or 2 Standards are measured ing 2 ul load up to 5 replicates Note software will guide the user with instructions in the large text box on the right Absorbance EE side of the screen The red Cursor X 725 light indicates the standard Absorbance 0 010 curve is incomplete and not o 650 675 725 750 ug ml O0 yet ready for sample 3 0 0 B1289 Wavelength nm measureme
39. d then the Hardware Wizard should appear and indicate it has located new hardware and will install the software for it If you are prompted whether or not to continue with the wizard select OK Note Windows XP and 2000 operating systems may give the message The software you are installing for this hardware OceanOptics USB2000 EEPROM Load has not passed Windows logo testing If you receive this message select continue anyway 5 Your NanoDrop ND 1000 Spectrophotometer should now be ready for operation If the software does not start properly you may need to configure the spectrometer Refer to the Troubleshooting section if this occurs 2 1 3 Configuring the System Font The NanoDrop software is designed to look best with the MS Sans Serif font 8 point To check that the system font is set to the proper selection 1 Open the Displays Properties by right clicking on the desktop and select Properties gt Appearance Additional step for Windows XP click on the Advanced button 2 From item list select icon 3 Select the MS Sans Serif western font and select 8 point size 4 Click OK NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Other selections can be used but may cause some text in the NanoDrop software window to not fit well or may cause the function selection tabs across the top to become inaccessible 2 1 4 Software Upgrades NanoDrop Technologies mak
40. data shown in archive files are represented as displayed on the software screen For both Nucleic Acid and Protein A280 samples data are normalized to a 1 0 cm 10 0 mm path For MicroArray UV Vis BCA Bradford and Cell Culture samples the data are normalized to a 0 1 cm 1 0 mm path For high absorbance UV Vis samples data are normalized to a 0 1mm path 8 4 D Sample Retention System Solvent Compatibility The NanoDrop ND 1000 Spectrophotometer is compatible with most solvents typically used in life science laboratories These include methanol ethanol n propanol isopropanol butanol acetone ether chloroform carbon tetrachloride DMSO DMF Acetonitrile THF toluene hexane benzene sodium hydroxide sodium hypochlorite bleach dilute HCI dilute HNO3 dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable 8 E Setting Up a Dymo 330 Label Writer Printer for Proper Operation To set DYMO default label to print with ND 1000 1 Open Control Panel then Printers and Faxes 2 Right click on Dymo printer then select Properties 3 Under General tab choose Printing Preferences then Advanced Then set the following e Paper Size 30256 e Halftoning Super Cell e Print Quality Barcodes and Graphics 49 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 Click OK to close this window then click Apply Next click on the Advanced
41. drophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension That occurrence can be overcome without 29 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurement 4 7 2 Special Cleaning Requirements for Proteins Proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form If this occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 20 times This will re condition the surface allowing for the liquid sample column to form 4 7 3 Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer using the regular Bradford assay unknown protein concentrations from 100ug ml up to several thousand micrograms ml ug ml can be determined The best linearity is in the 100 1000ug ml range A mini Bradford assay covers the approximate range of 15 to 125ug ml Coomassie dye dye and Coomassie dye protein aggregates are frequently encountered in Coomassie dye based protein assays With time particulate can be observed which can cause significant fluctuations in Absorbance readings It is also important to not
42. e automatic startup with MS Excel or other spreadsheet format see section below Examples of nucleic acid files are shown below 35 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 File Edit View Favorites Tools Help lt Back search Folders C AG GS X A EF Address Nucleic Acid Folders x 4 43 Multimedia Files aj Nucleic Acid 2003 09 09 ndt B NanoDrop Data 152 Nucleic Acid 2003 09 17 ndt H A administrator Nucleic Acid 2003 09 23 ndt B C Default Nucleic Acid 2003 10 03 ndt C BCA Protein Nucleic Acid 2003 10 14 ndt C Bradford Nucleic Acid 2003 10 27 ndt C Cell Culture Nucleic Acid 2003 11 06 ndt Microarray Nucleic Acid 2003 11 24 ndt Nucleic Acid 2 Nucleic Acid 2003 11 26 ndt C Protein A 280 Nucleic Acid 2003 12 01 ndt LIV Vis Nucleic Acid 2003 12 02 ndt LIV Vis HiAbs Nucleic Acid 2003 12 03 ndt E Jeff Nucleic Acid 2003 12 04 ndt E Leslie 5 Nucleic Acid 2003 12 05 ndt Philippe Nucleic Acid 2003 12 08 ndt BC Palm Nucleic Acid 2003 12 09 ndt Proqram Files 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 5 1 3 Archive File Format The files are in tab delimited format and should open in M
43. e the total analyte protein dye signal at 595nm is limited to 0 150 A as a result of the 1 0mm pathlength of the instrument the Bradford Coomassie dye reagent concentration and the acidic pH Making measurements in triplicate of Standards and Samples unknowns is good practice particularly with the limited assay signal obtained with the Bradford Assay Approximate Approximate Typical Reproducibility Lower Limit Upper Limit minimum 5 replicates ug ml ug ml SD ug ml CV m l sample range 100 500 ug ml 25 ug ml m l sample range 500 8000 ug ml 5 Mini sample range 15 50 ug ml 4 ug ml Bradford isum MES UIT sample range 50 125 ug ml 5 100 ug ml 8000 ug ml 4 7 4 Bradford Kits Protocols and Sample Preparation Commercial Bradford Protein kit manufacturers typically outline procedures for two different concentration ranges e A regular assay using a 50 1 reagent sample volume ratio To accurately prepare Standards we suggest using a minimum sample volume of 4 ul in 200 ul of Bradford reagent larger sample volume is preferable e A mini assay using a 1 1 reagent sample volume ratio To prepare sufficient volume of these 1 1 mixtures we suggest using a minimum of 10 ul of sample and 10 ul of Bradford reagent in a PCR tube Using the same pipettor for both volumes will eliminate any pipette to pipette accuracy differences In addition to the kit reagents protein standards e g BSA for generati
44. ear Perform the steps outlined in the Signal Error section of Troubleshooting 40 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 6 2 6 Low Detector Bias This occurs when the software has detected a problem with the spectrometer or the USB connection is lost while a measurement is being made If this message appears simultaneously with the Source Error message the USB connection has probably been lost To troubleshoot perform the steps outlined in the Signal Error section of Troubleshooting 6 2 7 Error 7 Occurred at New File This occurs when the C NanoDrop Data file folder has been removed from the C drive or it has been corrupted If this message appears click on the stop button in the error window Close the NanoDrop software Open Windows Explorer and create a new folder on the C drive Name the folder C NanoDrop Data case sensitive and close Windows Explorer Click on the arrow in the far upper left of the NanoDrop software Restart the NanoDrop software 6 2 8 ISR Not Installed This error is most likely caused by installing the software while the USB cable was connected to both the PC and instrument This will cause the OOlBase32 USB driver to not be properly linked To remedy this you must remove the OOI USB driver uninstall the operating software and then reinstall it Follow these steps to accomplish this 1 Plug the USB cable into the instrument a
45. ects the high concentration and utilizes the 0 2mm pathlength to calculate the absorbance Approximate Typical Reproducibility Upper Limit minimum 5 replicates ng ul SD ng ul CV 95 Detection Limit ng ul E sample range 1 5 100 ng ul 1 5 ng ul 2400 ssDNA sample range gt 100 ng ul 2 18 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 2 3 Unique Screen Features tido File Show Context Help NE A pon complete 9 1 6 2003 1 31 PM Exit 14 User Default Semple EDNEEWE Type Bisumpie 1230ngv _ i Batchindex 6 A230 Abs 10 512 A 260 10 mm path 2 431 A 280 10 mm path 1 321 2 M 260 230 EEN O iee RA do ze 3e do do do c5 cR 3o nsu 1215 5 3 0 0 A152 Wavelength nm 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 or Other for other nucleic acids The default is DNA 50 If other is selected the user can select an analysis constant between 15 150 See the Concentration Calculation Beer s Law Appendix for more details on this calculation and Abs the user selected wavelength and corresponding absorbance The wavelength can be selected by moving the cursor or using the up down arrows to the left of the wavelength box
46. ed or other decontaminating solution can be used to ensure that no biologically active material is present on the measurement pedestals The metal fiber optic fittings are made from 303 stainless steel and are very resistant to most common laboratory solvents see Solvent Compatibility appendix Special Cleaning Requirements for Proteins Proteins and solutions containing surfactants can un condition the measurement pedestal surfaces so that the liquid column does not form with 1ul samples If this occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 20 times This will re condition the surface allowing for the liquid sample column to form 3 1 3 Sample Size Requirements Although sample size is not critical it is critical that the liquid column be formed so that the gap between the upper and lower measurement pedestals is bridged with sample and the light path is completely covered by the sample Field experience indicates that the following volumes are sufficient to ensure reproducibility e Aqueous solutions of nucleic acids 1 ul Solutions containing significant amounts of dye 2 ul Purified protein 2 ul Bradford or BCA assay 2ul Aqueous solution of cellular material 1 ul It is best to use a precision pipettor 0 2 microliters with precision tips to assure that sufficient sample 1 2 microliters is used Lower precision pipettors 0 10 microliters and larger are not as good for
47. er limit of the vertical axis Sample Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 for RNA or Other for other nucleic acids The default is DNA 50 If other is selected the user can select an analysis constant between 15 150 See the Concentration Calculation Beer s Law Appendix for more details on this calculation and Abs the user selected wavelength black cursor and corresponding absorbance The wavelength can be selected by dragging the black cursor or using the up down arrows to the left of the wavelength box Note the user selected wavelength and absorbance are not utilized in any calculations Dye 1 user selected dye 1mm Dye 1 Abs measured absorbance of Dye 1 pmol ul Dye1 concentration based upon Dye 1 s extinction coefficient See the Concentration Calculation Beer s Law Appendix for more details on this calculation Dye 2 user selected dye 1mm Dye 2 Abs measured 1mm pathlength absorbance of Dye 2 pmol ul Dye2 concentration based upon Dye 2 s extinction coefficient See the Concentration Calculation Beer s Law Appendix for more details on this calculation ng ul concentration of nucleic acids in the sample based on absorbance at 260 nm and the nucleic acid analysis constant and normalized at 340 nm 260 280 ratio of sample absorbance at 260 and 280 nm The ratio of absorbance at 260 and 280 nm is used to assess
48. es periodic upgrades to the NanoDrop software These upgrades are available for download at www nanodrop com 2 2 Hardware 2 2 1 Cable Connections To make measurements with the instrument connect the USB cable to instrument and the PC plug in the 12V power supply and connect to the power input at the back of the instrument Note the power supply can remain plugged into the NanoDrop ND 1000 Spectrophotometer while the instrument is not in use When the unit is in this standby mode power consumption is 1 5 W and the flashlamp is not energized Also the instrument does not utilize a power switch or give a visual indication of the operability of the 12V power supply 2 3 Registering Your Instrument Please register your product We periodically update our software and add new features free of charge We would like to keep our user list updated so that we may alert you to these updates All information supplied to NanoDrop Technologies is completely confidential You can register at www nanodrop com NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 3 General Operation 3 1 The Sample Retention System 3 1 1 Basic Use The main steps for using the sample retention system are listed below 1 With the sampling arm open pipette the sample onto the lower measurement pedestal ES at TM gt Cet a E eS ton RA ec A SAA AS m ro A at EL S A PA AW Les I ee MX AA 3 2 Close t
49. ftware there are only three 3 general procedural steps to unknown protein concentration measurement The requisite order including generating the standard curve is as follows ne xj Step 1 Measure the File Edit Show Context He IT _m Reference BCA reagent 1 9 2004 3 30PM Exit a zero Standard Standard Curve User Default Ji 0 20 Max Absorbance Q Apply more standards J Reference The software will not allow measurement of Samples until at least 1 Standard and Replicate 4 Reference or 2 Standards ee are measured Note software will guide the peace ns bee to PEK user with instructions in the large text box on the right TA side of the screen The red B62nm 10 001 light indicates the standard Cursor X 725 curve is incomplete and not E EYT yet ready for sample 0 02 0 000 measurements 450 480 500 520 540 560 580 600 620 640 660 680 700 720 780 mg ml 0 000 3 0 0 B1289 Wavelength nm Ej y a E E S a 4 lt E E mi 2f NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 BCA Protein x Step 2 Measure Standards File Edit Show Context Help Lagi Pr tn ea 14972004 3 37PM_ exit Up to 5 replicates of each SS sr J Default standard can be measured J 1 00 Max Absorbance J Ready for Sample J Standard3 The software will not allow measurement of Samples until at least 1 Standard and Replicate 1 Reference or 2 Standards
50. gement module establishes whether User passwords will expire and if so when number of days Change password B Xx New password New password repeat gt 13 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 3 6 Utilities and Diagnostics This module is used to help troubleshoot operational problems with the instrument By selecting intensity check a characteristic spectrum is displayed that gives clues to the performance of the instrument For more information on using this module refer to the Troubleshooting section of this manual Diagnostics SO ep ey 14 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 3 7 4 Applications Modules 4 1 Functions Common to Each Application Module File Edit Show Context Help 4 1 1 Module Startup When the software starts you should see this message Click OK to Initialize the Spectrometer Load a water sample onto the pedestal first for best calibration CANCEL For best results ensure measurement pedestal surfaces are clean and load a water sample onto the lower measurement pedestal and then click OK After clicking OK the messages Initializing Spectrometer please wait and checking detector bias will appear When these messages disappear the instrument will be ready for use All data taken will automatically be logged in the appropriate archive file 4 1 2 Escape Key ESC The esca
51. gether 7 4 Warranty All spectrophotometers and accessories manufactured by NanoDrop Technologies are warranted against manufacturing defects in parts and labor for a period of one year A one year warranty extension may also be purchased 7 5 Recalibration 7 5 1 Wavelength This is auto calibrated each time the software is started No calibration is required by the user 7 5 2 Pathlength Accuracy Generally this is not required as field experience has shown that the path does not change appreciably even after several years of heavy use However it is a good idea to check the calibration every six months using CF 1 calibration fluid A calibration check procedure is available from the Download section at www nanodrop com 8 NanoDrop Technologies Contact Information Shipping Address Mailing Address 100 W Rockland Rd Suite I PO Box 415 Montchanin DE 19710 Rockland DE 19732 USA USA Voice 302 984 0334 Voice 302 984 0334 Fax 302 984 0336 Fax 302 984 0336 Email info nanodrop com Website http www nanodrop com NanoDrop is a registered trademark of NanoDrop Technologies Inc Other parties trademarks are the property of their respective owners and should be treated as such Copyright 2003 NanoDrop Technologies Inc 47 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Appendices 8 1 A Instrument Specifications Long Path Length 1mm Short Path Length for high concentration me
52. he liquid column is formed If necessary try 2 ul samples to ensure the column is formed Also proteins and solutions containing surfactants are known to un condition the measurement pedestal surfaces so that the liquid column does not form If this occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 20 times This will re condition the surface allowing for the liquid sample column to form Blanking with a sample instead When this occurs a non blank light intensity array will be used of the reference solution in place of the normal blank This can cause negative absorbances to be calculated If this occurs blank again with the reference solution and measurements should then be normal Last sample measured has dried In this case the dried sample resolubilizes when a water blank is on lower measurement pedestal pipetted onto the lower measurement pedestal which creates the and then a blank was made same effect of blanking with a sample Make sure both without cleaning the pedestal measurement pedestals are clean and blank again with reference first solution 43 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Sample is too dilute Measuring samples at or near the detection limit will result in measurements that can vary a significant amount Refer to the Measurement Concentration Range of the Application Module that you are using for the applica
53. he sampling arm and Y initiate a spectral measurement pper measurement Using the operating software on the pedestal PC The sample column is automatically drawn between the upper and lower measurement pedestals and the spectral measurement made lower measurement pedestal 3 When the measurement is complete open the sampling arm and wipe the sample from both the upper and lower pedestals using a soft laboratory wipe Simple wiping prevents sample carryover in successive measurements for samples varying by more than 1000 fold in concentration See www nanodrop com for performance data on sample carryover 3 1 2 Cleaning the Sample Retention System Upon completion of each sample measurement wiping the sample from the upper and lower pedestals as shown above is sufficient to prevent sample carryover and avoid residue buildup NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 However after measuring a large number of samples it may be necessary to clean the areas around the upper and lower pedestals thoroughly This will prevent the wiping after each measurement from carrying previous samples onto the measurement pedestals and affecting low level measurement A final cleaning of all surfaces with de ionized water is recommended prior to storing the instrument Decontamination of Measurement Pedestals If decontamination is necessary a 5 25 solution of sodium hypochlorite bleach freshly prepar
54. her sample containment devices and allows for clean up in seconds In addition the ND 1000 has the capability to measure highly concentrated samples without dilution 75X higher concentration than the samples measured by a standard cuvette spectrophotometer 1 2 Operation A 1 ul sample is pipetted onto the end of a fiber optic cable the receiving fiber A second fiber optic cable the source fiber is then brought into contact with the liquid sample causing the liquid to bridge the gap between the fiber optic ends The gap is controlled to a 1mm path A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light after passing through the sample The instrument is controlled by special software run from a PC and the data is logged in an archive file on the PC 1 3 Applications UV VIS spectrophotometry is simple for samples as small as 1 ul using the NanoDrop ND 1000 Spectrophotometer The small sample requirement and ease of use make the NanoDrop ND 1000 Spectrophotometer ideally suited for measuring e Nucleic acid concentration and quality of nucleic acid samples up to 3700 ng ul dsDNA without dilution Fluorescent dye labeling density of nucleic acid microarray samples Purified protein analysis A280 up to 100 mg ml BSA Bradford Assay analysis of protein BCA Assay analysis of protein Cell density measurements General UV Vis spectrophotometry NanoDrop ND 1000 Spectr
55. icrosoft Excel or an equivalent spreadsheet program Once the data has been opened in Excel the result should be similar to that below The first row is a header that identifies the data in the columns The columns beginning with N and extending to the right are the absorbances at the wavelengths shown in the header row Ed Microsoft Excel E xl File Edit View Insert Format Tools Data Window Help Acrobat OSHA GRY BB o r 5 M gt ara 10 BZU EFS3H 8 tE 5 A 22 A26 Y E Nucleic Acid 2003 11 20 ndt E Bl x BH Bl EII H EA E AN STET d Sample ID Date Time ng ul 250 280 260 230 Constant Cursor Po Cursor ab Serial Firmware Software Config 220 2 2 7 30ng ul DNA 11207200 12 17 PM 30 64 72 0 93 50 230 0 658 USB2E55 USB2000 3_Beta 1 003099 0 695 D 6 3 3U0ng ul DNA 11 20 200 12 18 PM 29 54 1 82 0 94 50 230 0 626 USB2bE55 USB2000 3 Beta 1 003099 0 93 0 8 4 350ng ul DNA 11 20 200 12 18 PM 326 85 1 84 1 82 50 230 3 586 USB2E55 USB2000 3 Beta 1 003099 4 534 4 3 5 350ng ul DNA 11 20 200 12 20 PM 324 43 1 84 1 83 50 230 3 54 USB2E55 USB2000 3 Beta 1 003099 4 48 4 3 The data may be edited and or reformatted and stored under names of the user s choice The spectrum can be re plotted from the wavelength data if needed for further analysis Note1 absorbance data shown in archive files are represented as they are displayed on the screen For Nucleic Acids and Protein A280 Application modules data
56. ime Sample Type choices are Reference Standards 1 5 and Sample The software will guide you to measure your Reference then at least one Standard before allowing measurement of samples Replicate counter for tracking replicate number during Reference and Standard measurement 26 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Reset Window F11 clears all replicates of all standards Reset This Std F12 clears all replicates of the selected standard Absorbance at 562nm the Cu BCA complex s absorbance at 562 nm Cursor and Absorbance this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance The cursor wavelength can only be selected by dragging the cursor Note the user selected wavelength and absorbance are not utilized in any calculations mg ml the concentration of the sample unknown 4 6 6 Making BCA Measurements In keeping with kit manufacturers guidelines generation of a Standard Curve is required every time the BCA Assay is run Single or Multi point standard curve generation is incorporated into the software A Standard Curve can be developed using a Reference BCA reagent only no protein and a single replicate of one Standard The multi point standard curve generator allows a maximum of five 5 replicates for each of five 5 different Standards There is no set order in which Standards must be run Using NanoDrop version 3 0 so
57. ions using the up or down arrow keys located to the left of the sample type box A description of each sample type is given below SampleType A general reference setting based on a 0 1 1 mg ml E ene protein solution producing an Absorbance at 280 nm of 1 0 A where the pathlength is 10 mm or 1 cm La Le 24 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 leT Bovine Serum Albumin reference Unknown sample alo ae d ad protein concentrations are calculated using the mass ABSA extinction coefficient of 6 7 at 280 nm for a 1 10 mg ml BSA solution sample Type IgG reference Unknown sample protein concentrations lr G are calculated using the mass extinction coefficient of 119 13 7 at 280 nm for a 1 10 mg ml IgG solution Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution sample Type REESE Lysozyme sample Type Other protein E amp MW User entered values for molar extinction coefficient M cm and molecular weight MW in kilo Daltons for their e x1000 50 00 respective protein reference Maximum value for e is 999 X 1000 and maximum value for M W is 9999 X M W kDa 50 00 1000 sample Type ell the User entered mass extinction coefficient L gm cm for o a 10 mg ml 1 solution of the respective reference
58. ith V3 0 0 will be present when running V3 0 1 If the accounts are not available then passwords log and user preferences log files must be manually copied from the c program files NanoDrop 3 0 0 folder to the c program files NanoDrop 3 0 1 folder Administrator privileges on the pc will be required for this 3 4 User Preferences Each respective user can select settings they most commonly use for four of the Application modules e Nucleic Acids e MicroArray 12 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 e UV Vis e Protein A280 ETT x File Show Context Help User Default aa Nucleic Acids Microarray Protein A280 DNA 50 DNA 50 Sample Type 1 Abs 1 mg mL x UV Vis A1 J300 HiAbs X on AZ jj roo Normalize On Once the settings are saved they will be automatically opened when the respective user is logged in to the NanoDrop software and any of the four Application modules are open Note user preferences are stored in a log file When upgrading to a newer version of the software this file should be preserved If after upgrading to a new software version the user preferences do not appear correctly the log file should be manually copied to the proper directory See Passwords log section for more detail 3 5 Change Password This module enables each user to change their respective password Note the Administrator using the Options entry in the Account Mana
59. l BCA Protein kit manufacturers typically outline procedures for two different protein concentration ranges e A regular assay using a 20 1 reagent sample volume ratio To accurately prepare Standards we suggest using a minimum sample volume of 4 ul in 80 ul of BCA reagent larger sample volume is preferable e Amini assay using a 1 1 reagent sample volume ratio To prepare sufficient volume of these 1 1 mixtures we suggest using a minimum of 10 ul of sample and 10 ul of BCA reagent in a PCR tube Using the same pipettor for both volumes will eliminate any pipette to pipette accuracy differences Note If you run the assay at 60 C doubling the volumes may afford greater insurance against skewed results from evaporation condensation within the sealed reaction tube In addition to the kit reagents protein standards BSA for generating a Standard Curve are also provided for the BCA method by the manufacturer Follow the manufacturer s protocol for the assay including recommended incubation times and temperature Additionally use the respective Standard e g BSA and dilutions that cover the analytical mg ml range of interest Note since the ND 1000 can measure higher protein concentrations you may need to supply your own protein standards at higher concentrations than provided by the manufacturer 4 6 5 Unique Screen Features View Standard Curve F8 selecting this button allows the user to view the Standard Curve at any t
60. m Ho E EID xl BEgES LL ix teg T I Flash Delay Sicbe Lamp Conect i poz po lt img x Bosa H pai LE A al Electrical Dark Spectrometer Configuration E xi Wavelength Calibration A D Interface Reference Monitoring Stray Light Correction Detector Linearity Spectrometer Type s2000 PC2000 U582000 HF2000 A D Converter Type us2000 USB Serial Number MEF Em Cancel Apply Help 5 Confirm Power Supply Operation To confirm that the 12V power supply is working properly connect the leads of a volt ohmmeter to the outlet of the supply The voltage should be 12 20 Vdc center positive Confirm that Light is Reaching the Sample Open the Utilities and Diagnostics module of the software Select OK to initialize the spectrometer You should see a detector bias value greater than 65 This indicates that the PC and instrument are communicating properly With the sampling arm down select Intensity Check This should generate an output similar to the image in step 3 If no output is present the flashlamp may have failed If there is a spectrum present this indicates that the flashlamp is operating In this case one of the fiber optic cables might be broken reducing the amount of light that reaches the spectrometer Confirm Spectrometer Operation Open the Utilities and Diagnostics module of the software Select OK to initialize the spectrometer You should see a detector bias value greater tha
61. n 65 39 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 This indicates that the PC and instrument are communicating properly To confirm that the spectrometer is functioning open the sampling arm and shine a flashlight directly into the lower measurement pedestal or place very near a fluorescent light If the spectrometer is operating properly the detector bias value will increase significantly 8 If none of the troubleshooting steps above solve the problem refer to the Technical Service section for getting help from your distributor or NanoDrop Technologies 6 2 Other Software Error Messages 6 2 1 Error USB2000 Unable to find device with serial This message appears when communication has been lost between the PC and instrument This usually occurs due to inadvertent disconnection of the USB cable In most cases reconnecting the USB cable will re establish the connection With Windows 98 and ME it may be necessary to restart the computer to re establish the connection This message will also appear if the software was installed while the instrument was connected to the PC via the USB cable In this case perform the procedure in the ISR Not Installed section of Troubleshooting to remedy this 6 2 2 Can t find file OOIDRV INI This error occurs when trying to install the software without administrator privileges Contact your system administrator to install the software 6 2 3 An Error
62. naninenanons 18 4 2 2 Measurement Concentration Range ccoooccccconccoconinoconnnoonnnnonnnononononononononononaninnaninonaninnns 18 4 2 3 UNIQUE SCTECI FAS een eee Gh ea aes ee hee 19 4 2 4 Spectrum Normalization occooocccccocnnococonocononoconnonononononononannnnononononononannonanononaninenaninnns 20 4 3 MICROARRAY MEASUREMENT MODULE cccseecccseececsecccceececeuececueceesceesuecessueeesseeessneeesaeeesanes 20 4 3 1 Sample Volume Requirements cccoonncccccnnocononooconocononononononanononnnnonononononononarononannonanonenaness 20 4 3 2 Measurement Concentration Range ccoooncccoconcnoconinoconnnoonnnnonnnonononononononononononinnaninonaninnos 20 4 3 3 Unique Screen Fealures oocccoconococonococconocononoconnonononononononanonnnnnnnnnnnnononnononononananonarinenaninnns 21 434 Baselhe CalCulaon oit eis eb a A A E desee esc weaned 22 4 3 5 Fluorescent Dye SelectiON cocoocccccocononecccnnoncnononoonanonononnnnnnonanonnonanonnnnnnnnnnnnnnnnenanons 22 44 UV VIS MEASUREMENT MODULE 0 00 eoe ex aedis adu peo qp es ade uu te manioemeacieael maces 22 4 4 1 Sample Volume Requirements cccoocncccccnnccoconoocnnocononononononanononannonononononononanononannonanonenanoss 22 4 4 2 Measurement Concentration Range ccococcccconcnoconinoconnnconnnonononnononononononononononnnnaninonaninnns 22 4 4 3 UNIQUE ScIeembealllles au ea ttp pt eio sies ti lc 23 49 EBOIENAZOGU MOD UE sad 23 4 5 1 Sample Volume Requireme
63. nd the PC 2 Select the following path to bring up the System Properties screen Start Settings gt Control Panel 2 System 3 Onceatthe System Properties screen For Windows 2000 and XP select the Hardware tab and then click on Device Manager For Windows 98 and ME select the Device Manager tab 4 Atthe bottom of the screen of the Device Manager page click on the that is to the left of the Universal Serial Bus controllers line 5 Find the Ocean Optics USB2000 EEPROM Load device driver highlight it and then delete it 6 Unplug the USB cable from the instrument 7 Uninstall the OOlBase32 software To do this select the following path and follow the onscreen instructions Start Settings gt Control Panel gt Add Remove Programs 8 Restart computer 9 Reinstall the NanoDrop software 10 Restart computer again 11 After computer has rebooted reconnect the USB cable to the instrument and wait 1 minute 12 Start NanoDrop software System should operate normally 6 2 9 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 NanoDrop software using the installation CD or downloaded from www nanodrop com 6 2 10 EZUSB SYS Cannot Be Found If this error message appears do the following depending on your operating system Windows 2000 type C WINNT in the file path text box If the file ooi usb inf cann
64. ng refer to the Technical Service section for instructions on how to contact NanoDrop Technologies and what information must be sent to allow for diagnosing the problem 6 5 Technical Service If after referring to the above troubleshooting tips you are unable to resolve your problem please contact your local distributor or NanoDrop Technologies for help The following information will be very helpful 1 JPG image of Utilities and Diagnostics module To get this open this module and select OK to initialize the module Select Intensity Check Once the spectrum has been created choose File gt Save Window as shown below Save to your hard drive and email as an attachment to your distributor or to info nanodrop com Diagnostics xj Show Context Help Page Setup Print ad Ctrl P Detector Bias cm c Ea Exit Ctr Q TU 3500 0 3000 0 Y V 2500 0 A M ul u mi 1500 0 1000 0 few we INN a bus i 500 07 9 m LA car 0 0 EI I I L I I I I 1 I L I 230 0 300 0 350 0 400 0 450 0 500 0 550 0 600 0 650 0 700 0 750 0 Wavelength nm Serial Number Configuration USB2G1212 B1154 11 002097 0 22 64 24 45 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 2 Application Module Screen Captures Screen captures of the actual spectrum as seen on your PC of great use in diagnosing problems Making a screen capture is quite easy When in an Application Module press Alt Print
65. ng a Standard Curve are provided by the manufacturer Follow the manufacturer s recommendation using Standard BSA dilutions that cover the analytical ug ml range of interest Note since the ND 1000 Spectrophotometer can measure higher protein concentrations you may need to supply your own protein standards at higher concentrations than provided by the manufacturer 4 7 5 Unique Screen Features View Standard Curve F8 selecting this button allows the user to view the Standard Curve at any time 30 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Sample Type choices are Reference Standards 1 5 and Sample The software will guide you to measure your Reference then at least one Standard before allowing measurement of samples Replicate counter for tracking replicate number during Reference and Standard measurement Reset Window F11 clears all replicates of all standards Reset This Std F12 clears all replicates of the selected standard Absorbance at 595nm protein dye complex s absorbance at 595 nm Cursor and Absorbance this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance The cursor wavelength can only be selected by dragging the cursor Note the user selected wavelength and absorbance are not utilized in any calculations ug ml concentration of the sample unknown 4 7 6 Making Bradford Protein Measurements In keeping with kit manufa
66. ng else Level 5 is the suggested level for a New User account While the level can be set to 10 that isn t recommended since that would give another user full access to the Account Management module 3 3 3 Account Log in 1 The user may log into his account by clicking on the down arrow next to the User name on the User Default hd l main menu 2 This will bring up the enter password dialogue x box below Enter the valid password User ID red Password 11 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 E Timed dialog Note The specific user account fred in the example above will automatically revert back to the Default User after 4 hours System idle timeout Returning to main menu Click If the user is using any of the Application modules a screen Cancel to stay in this module indicating that the time is about to expire with a 30 second countdown will be displayed If the user elects CANCEL the clock with re set and the respective user account and Application will remain active If the time expires the open Application module will close returning to the Main Menu and the Default user Auto Shutdown in 29 Seconds 3 3 4 Account Log out The respective user s account will remain active until either the software System idle timeout is exceeded or the user logs out The latter may be done by clicking on Default next
67. nnns 30 4 7 4 Bradford Kits Protocols and Sample PreparatiON ooccccocncccccnnocccnnoccnnonononononononaninnns 30 4 7 5 EniguesScreen Features eins 30 4 7 6 Making Bradford Protein Measurements occcoooncncocnnoconinoconnnooonnnnonnnonononnonnnononononaninonaninnos 31 4 7 7 Standard Curve teastules ia 32 4 7 8 Exiting the Bradford Module lid 33 HO GELECUETURE NORUNT m 33 4 8 1 Sample Size Requirements coooccccoccncccoconocononoconnonononononononanononnnnononononnnnononononarononaninenanens 33 4 8 2 Cell suspension Concentrations oooccccconnocononecononocononenonononcnnononnnonnnnononononanononaninenaninnns 34 4 8 3 Unique Screen Features cccoocccccccnnccoconocononocononcononononononanononnnnnnonnnononnnnnnononanononaronenaninnnans 34 4 8 4 Sample Homogeneity ccccoooncncocncccocnnocononononononononoonnnononnononnnononnnononnnnnnnnnonnnononinonaninnnannnnnns 34 ATCHIVCA Dala cta H 35 5 1 1 Data Storage Foral sate He 35 Dees o Fe CIC QT OI sm tsk oat pata i sae b LIU LL eae ee a ee LEE 35 DAs ACV FIIS TOM otre am a Dao vsu a vast oops catia aeu toa too Lone eer 36 5 1 4 Automatically Opening Archive Files with MS Excel ooocccoconicoccccnococincocnnoconononononononoss 37 TrOUDIEeSHOO TINO iria ios 38 GU O E et I ESL NM A MD Ex dad 38 2 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 0 2 OTHER SOFTWARE ERROR MESSAGES sui es a 40 6 2 1 Error USB2000 Un
68. nts cccoocnnccccnnococonocconocononononononanononannonononononononanononannonaronenanons 23 4 5 2 Special Cleaning Requirements for Proteins ossis seen 24 4 5 3 Measurement Concentration Range cococcccconccocononoconnnconnnnononononononononononononaninnaninonaninnns 24 4 5 4 Unique Screen Features E Li a EEEa RAAN icu T OPUS 24 4 5 5 Spectrum Normalization ata 25 C9 PROTEIN BCA MODULO a 25 4 6 1 Sample Volume Requirements cccoocncccccnnococonoocnnoconononononononononannonononononononarononannonarinenanons 26 4 6 2 Special Cleaning Requirements for ProteinS coooccccoccnccccononoocnnononononononnonononanononaninnos 26 4 6 3 Measurement Concentration Range ccooocccccoccnoconinoconinconnnnonnnononononononononononannnnaninonaninnos 26 4 6 4 BCA Kits Protocols and Sample Preparation oooccccoccccocnnccccnnocononononononononononnonaninnos 26 4 6 5 NIQUES Screem Fealllles cio uy A A 26 46 6 Making BCA Measurements aici Rea ie a ee NN 27 AGS Stahoard QGurve Fealtuless it 28 46 0 EXN Me BCA MOS A 29 do PROTEIN BRADFORDIMODUEE aa ideada 29 4 7 1 Sample Volume RequireMeNt cooocccocnnccocnniccnnccncncnononcccnoncnnnonccnnnnonnncnonancncnancnnnnnnnnnannnnannnns 29 4 7 2 Special Cleaning Requirements for ProteinS coooccccccnincccnnococonononononononconononanononaninnos 30 4 7 3 Measurement Concentration RAange ccconcccconccoconinoconnnnonnononnnnnonononononononononannnnaninonani
69. nts y I E a a S A a lt E E 31 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 Protein Bradford E X File Edit Show Context Help pe Re blank Measur A 0 20 Max Absorbance Print Screen Start Report 11 9 2004 3 50 PM Standard Curve User Default J Standard 4 Y Ready for Sample Replicate zal Std ug ml 1000 Using 2 ul load up to 5 replicates of your4th standard and measure It is best to blank between standards Absorbance 595 nm Cursor A 725 Absorbance 0 013 79 ug ml 1000 0 172 650 675 3 0 0 B1289 Wavelength nm Protein Bradford m l x File Edit Show Context Help Re blank 11 9 2004 4 13 PM Standard Curve User Default 9f 0 20 Max Absorbance J Sample Sample 200ug ml BSA ID Batch Index 0 Y Ready for Sample Load your 2 ul of your sample and click the measure button u E o o A Bel lt Absorbance 2 595 nm Cursor X 725 Absorbance 0 013 136 0 068 1 1 I 1 1 650 675 750 Wavelength nm 3 0 0 B1289 gnat 4 7 7 Standard Curve Features rev 16 Apr 04 Step 2 Measure Standards Up to 5 replicates of each standard can be measured The software will not allow measurement of Samples until at least 1 Standard and Reference or 2 Standards are measured If erroneous or non representative readings are encountered for a specific standard all
70. o shut down 6 3 Hardware 6 3 1 Chattering Solenoid This phenomenon is characterized by a very rapid chattering not a distinctive clicking sound If this occurs it is caused by a malfunctioning component on the circuit board If this occurs contact your local distributor or NanoDrop Technologies for service 6 3 2 Intermittent USB Connection Failure Unusual performance that occurs intermittently is very hard to diagnose In some cases this can be caused by a faulty USB board in the PC If your instrument is exhibiting strange performance that is not addressed by any of the other troubleshooting tips it would be helpful to install the software and operate the instrument on another computer to see if the behavior is duplicated A list of known USB problems is listed below Possible Cause Possible Solution Static Electricity from the user Use a grounding wrist strap 42 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 PC has gone into standby or Change the PC s power 21x hibernate mode management properties SO Power Schemes Alarms Power Meter Advanced Hibemate that it does not go to WA omnes Noi fat chengrd he eio bebo el odi Standby or Hibernate mode is P Power schemes when plugged in To reach the power management Save As Delete screen choose Start gt ES Control Panel gt Power r Settings for Home Office Desk power scheme When computer is 1
71. oDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 For a more detailed description of each module refer to section 4 3 3 Account Management The Account Management module provides options for directing where specific data files are archived for users desiring to segregate their data into a specific folder It allows existing NanoDrop ND 1000 Spectrophotometer users familiar with the existing archived data files to operate as accustomed enabling immediate access to and running of the software The Account Management module is accessible to the Administrator only The Change Password module can be accessed by any user having an authorized account ID 3 3 1 Setting up Accounts User Default administrator At the time of version 3 0 1 software installation there are two specified users in the NanoDrop software Others may be added see Administrator below Administrator the Administer has full access to all account settings and the highest security level in the NanoDrop software level 10 At the time of software installation only the Administrator can access the Account Management module Through that software module the Administrator can e Add New Users e Modify a User e Delete a User e Set conditions for the accounts using the Options entry The respective parameters within the Options menu are self explanatory See examples of screens below User Access Manager 1 i X Actions OT cer Delete Use
72. ophotometer User s Manual V3 0 1 rev 16 Apr 04 2 Initial Set Up 2 1 Software 2 1 1 Computer Requirements The NanoDrop software will only run on an IBM compatible PC meeting the below criteria No Mac versions of the software are available e Microsoft Windows 98 Millennium Edition XP or 2000 operating system The operating software will not work with Windows 95 or Windows NT 233 MHz or higher processor CD ROM drive 32 MB or more of RAM 40 MB of free hard disk space Open USB port the instrument can only be connected via the USB port Microsoft Excel or other spreadsheet program to manipulate archived data optional 2 1 2 Installation WARNING The system software must be loaded onto the PC before the USB cable is connected Administrator access on the PC is required to install the software To properly install NanoDrop software 1 Close all programs and make sure that the USB cable is unplugged 2 Insert the operating software CD in the CD drive of the PC The software 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 Install Once the software menu is available choose Install Software and follow the onscreen prompts Restart your pc when prompted 3 Remove warning label from back of the instrument and connect the USB cable to the PC and to the instrument 4 There will be a delay of approx 1 minute an
73. ot be found type C 1WINNTUNF in the file path text box Windows 98 ME type C WINDOWSI SYSTEM in the file path text box If the file ooi usb inf cannot be found type C WINDOWSIINF in the file path text box 41 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Windows XP type C WINDOWS in the file path text box If the file ooi_usb inf cannot be found type C WINDOWSIINF in the file path text box This should allow the installation to complete successfully 6 2 11 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 on the computer Contact your system administrator if this occurs 6 2 12 Error 8 Occurred at Open File This error message occurs when the user attempts to take a measurement while the data file is open Close the data file and you should be able to continue taking samples normally 6 2 13 Insufficient Memory This error message or others with similar wording occurs when attempting to install the operating software on a computer that does not have at least 40MB of free hard disk space 6 2 14 Error Code 31 This error appears when attempting to print when a printer is not attached to the PC It is non fatal and will not cause the software t
74. pe key is set to exit out of all screens Hitting the escape key twice will log the user out of an application module 4 1 3 Measure F1 Each time a software module is opened initiated the Measure button is inactive as noted by its grayed out appearance A Blank must first be measured before the Measure button will become active The Measure button is used to initiate the measurement sequence for all samples non Blanks It is actuated by depressing the F1 key or clicking the Measure button The entire measurement cycle takes approximately 10 seconds 4 1 4 Blank F3 Before making a sample measurement a blank must be measured and stored see the Appendix Blanking and Absorbance Calculations for more details on absorbance calculations After making an initial blank measurement a straight line will appear on the screen subsequent blanks will produce a nearly straight line that is very near zero see images below 15 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 File Edt Show Context Help fisnis 2003 458 PM Exit User Detault i peca monaco 12 16 2003 3 38 PM Exit User Default Sample Naso z ID Batch index 0 Sample iD nick Index A 260 10 mem path 0 000 ASOD 10 men path om0 260 200 Nan ED 230 HoN wis Zn zo slo xh xe ib e mo xb sh x xb su xe nb Qu Wanelenagth nm 3 0 0 COUT A 260 10 mm path 0008 A 2010 mm p
75. r Options ENS All Llsers Full Mame Level Active Locked Expired Expires ah Not Locked Nat Expired Fred act e Mot Locked Mot Expired Never lef TS ave Notlocked Not Sres 10 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 CInIONS ET x j Edit the current user parameters and click Continue to save or Cancel to exit without saving Maximum Password Attempts Active D lt UserID fred Minimum Password Length uu DIENEN Password Expiration enabled Password Password I I o Expiration Days Security level 5 Unlock account and or reset expiration CO NTI FJ LI E C AN C E L Note the initial Administrator s password is nanodrop It is strongly recommended that the Administrator change this password after initial account set up Default User the Default setting enables any user to access all the active software measurement modules It is not password protected All data generated will be automatically archived into the Default folder within the NanoDrop data folder Within the Default Folder are subfolders labeled with the respective name of the NanoDrop Application module used for measurement Note the Default user option is always functional even if all specific user accounts including the Administrator s become locked 3 3 2 Account Levels There are only two security levels in the current NanoDrop software level 10 and everythi
76. replicates of that standard are cleared by selecting Reset this Std F12 Additionally all Standards can be deleted at once using the Reset Window F11 button Step 3 Measure Sample Once a minimum Standard curve has been established the red indicator light will turn green allowing the user to either start measuring samples Sample concentrations are calculated using linear interpolation point to point between the two Standards flanking the unknown Sample Note In order to obtain a concentration value ug ml the sample unknown must fall within the limits of the Standard curve selecting this button allows the user to view the Standard Curve at any time The respective Standard Curve can be printed but not saved for later use since its utility is limited to the specific time period in which it and the respective unknowns are assayed 32 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Protein Bradford Std Curve E xj oes Abet Abs User Default Reference 0 joo Standard 1 10 224 Standard 2 10209 Standard 3 loia 3 0 0 A152 ray ar ars err ext reni i M Page Standard 5 0 065 ide 0 24 11 6 2003 12 36 i i Regular Bradford curve covers 200 029 pH 8000 ug ml Note the linear range is pr gr 100 1000 ug ml 0 16 0 14 E 0 12 0 10 0 08
77. return to the specific Application module 4 1 10 Sample ID 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 This makes the software barcode reader compatible 4 1 11 Batch Index The Batch Index indicator is activated when a Sample Report is being recorded It indicates the sample number of the last sample processed in the current batch and increments with each successive measurement until the Sample Report is fully populated The next sample measured 17 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 after the current Sample Report is complete will automatically reset the Batch Index number clear the entries in the Sample Report and will begin to populate the next Sample Report 4 1 12 Printing via the Print Menu A Print dialogue can be initiated from the File pull down menu accessible within all modules Nucleic Acids Edit Show Context Help Ec Setup Print Window 4 Ctrl F Exit Crea The Print Window option and Ctrl P enable selection of printers available to the respective user 4 1 13 Exit The command for closing all Applications and supporting options After clicking the Exit button
78. s occurs properly then all the user defined dyes that were created with V3 0 0 will be present when running V3 0 1 If the user defined dyes are not available then the dye list log file must be manually copied from the c program files NanoDrop 3 0 0 folder to the c program files NanoDrop 3 0 1 folder Administrator privileges on the pc will be required for this 4 4 UV VIS Measurement Module The UV VIS Absorbance tab allows the NanoDrop ND 1000 Spectrophotometer to function as a conventional spectrophotometer Sample absorbance is displayed on the screen from 220 nm to 750 nm and cursors permit the measurement of individual peaks 4 4 1 Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring most aqueous samples If you are unsure about your sample composition or your pipettor accuracy a 1 5 2ul sample is recommended to ensure that the liquid sample column is formed and the light path completely covered by sample 4 4 2 Measurement Concentration Range All ND 1000 spectrophotometers will measure absorbances up to the 10mm pathlength equivalent of 15 A ND 1000s with serial number gt 500 or that have been field retrofitted can utilize the short path length 0 2mm which enables the 10mm pathlength equivalent of 75 A 22 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 Recording 12 5 2003 10 08 AM Exit
79. spectively without dilution A table of sample concentration ranges is listed below Detection Approximate Typical Reproducibility Sample Type Limit Upper Limit minimum 5 replicates pmol ul pmol ul SD pmol ul CV Cy3 Cy3 5 Alexa Fluor 555 and Alexa Fluor 660 Cy5 Cy5 5 and Alexa Fluor 488 and Alexa Fluor EN 594 Alexa Fluor 546 sample range 0 20 4 0 pmol ul 0 20 pmol ul sample range gt 4 0 pmol ul 296 sample range 0 12 2 4 pmol ul 0 12 pmol ul sample range gt 2 4 pmol ul 2 E sample range 0 40 8 0 pmol ul 0 40 pmol ul sample range gt 8 0 pmol ul 296 sample range 0 30 6 0 pmol ul 0 30 pmol ul sample range 76 0 pmol ul 2 20 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 3 3 Unique Screen Features MicroArray E xj File Edit Show Context Help p Re btank Print Screen Start Report 1 9 2004 2 53PM Exit Measure Measurement User Default A 0 25 Max Absorbance Sample Type DNA 50 Sample Alexa 555 6 fold ID dilution H20 Batch Index o X 260 ImmAbs 0 041 Dye1 Alexa Fluor555 5 Dye 1 Abs 0 150 pmol ul Dye 1 10 0 Dye2 Alexa Fluor 647 Dye 2 Abs 0 001 pmol ul Dye 2 0 0 260 280 122 0 03 1 1 1 I 1 i 1 n i 220 250 300 350 400 450 500 550 650 700 750 ng ul 17 0 3 0 0 B1312 Wavelength nm Y imm Absorbance i B l iM Max Absorbance used to rescale the upp
80. the user has 10 seconds to cancel the exit command If no action is taken after 10 seconds the exit command is carried out Note that all measurement data is automatically saved to an archive file and requires no user action 4 1 14 Context Help Context Help is enabled in the Main Menu all function modules and the Application modules The help feature is enabled by choosing Show Context Help from the Help menu or by selecting Ctrl H Once enabled placing the cursor on elements of the screen will automatically generate an explanation of that element Context Help remains active until the user deselects it 4 2 Nucleic Acid Measurement Module Nucleic acid samples can be readily checked for concentration and quality using the NanoDrop ND 1000 Spectrophotometer To measure nucleic acid samples select the Nucleic Acid Application module 4 2 1 Sample Volume Requirements Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous nucleic acid samples However if you are unsure about your sample or your pipettor accuracy a 1 5 2ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample 4 2 2 Measurement Concentration Range The NanoDrop ND 1000 Spectrophotometer will accurately measure nucleic acid samples up to 3700 ng ul without dilution To do this the instrument automatically det
81. the PC Then reinstall the NanoDrop software refer to section 2 1 2 Reset the Spectrometer Configuration Occasionally the software driver used to control the USB communication must be reset manually To do this perform the steps below 1 Start the OOIBase32 software Start gt Programs gt Ocean Optics gt OO Base32 gt OOIBase32 If you received the message This appears to be the first time choose default and select OK Select the following file c program fileslocean optics ooibase32 default spec Once the software starts select the Spectrometer menu and select Configure from the dropdown menu 2 In the Spectrometer Configuration window select the A D interface tab and choose the settings as shown below right Even if the USB Serial Number slot is already filled with the proper serial number use the pull down menu to select the serial number This will enable the Apply button at the bottom of the screen Select Apply and then select OK The spectrometer should now be configured properly Shut down the OOlBase32 software and restart the PC If no USB serial number is available as a choice in the dropdown menu close the OOIBase32 software and restart the PC Then open OOlBase32 again and configure the A D interface again as outlined above DITE S ESTE ES o a ER 00 E Fie Edi Wer Crerlar Spectrometer Speck Tere Acquedion Script Wind Help DG EB mm
82. the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm See 260 280 Ratio section of the Troubleshooting section for more details on factors that can affect this ratio 21 NanoDrop ND 1000 Spectrophotometer User s Manual V3 0 1 rev 16 Apr 04 4 3 4 Baseline Calculation The software will automatically calculate a baseline between 400 and 700 nm The green vertical line on the screen represents the peak wavelength position for Dye 1 and the red vertical line represents the peak wavelength position for Dye 2 4 3 5 Fluorescent Dye Selection Eight additional fluorescent dyes have been added to the original Cy3 and Cy5 cyanine dyes They can be selected using the scroll arrows or by highlighting the respective Dye 1 and Dye 2 boxes The respective Absorbance excitation wavelength and extinction coefficient will automatically be utilized for measurement and subsequent concentration calculation of the respective dye e Cyd Cyb Alexa Fluor 466 Alexa Fluor 546 Alexa Fluor 555 Alexa Fluor 594 Alexa Fluor b4 Alexa Fluor 660 Cy3 5 Cy5 5 Note If upgrading to V3 0 1 from V3 0 0 the dye list log file should be automatically copied to the proper directory for use with V3 0 1 If thi

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