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Agilent 2100 Bioanalyzer User's Guide for

1. ssesscssessesseesessessessesseeneees 36 Changing the View of the Results c cccccccsessessessessesseesessesseesesseesessecseesssusssesneeeesnsseneeseanens 52 Whe Results Walle cece cts ce cescte seriseronsee e E EE 61 Data Analysis RNA Cy5 Labeled Nucleic Acids and RNA Smear Assay 66 How the Agilent 2100 Bioanalyzer Software Analyzes Data c cccccescssssssestessesseetesseseeeees 66 Changing Your Data Analysis RNA and RNA Smear AssayS s scessccsessessessesseesesseeseeneees 69 Changing the View of the Results c cccccccsessessessessessessecsesseesesseescssssseesssuesuesneessnssnsseeeeaees 85 The Results Table Data Analysis Protein Changing Your Data Analysis Protein Changing the View of the Results The Results Table Data Evaluation sctascssiccsctscnsicesscsvarssccecacevanescsvacttanssccovevseetacesceverarad setscvenesisontartvssaccravereuadtccie Starting the Data Evaluation Tool Loading Electropherograms in the Data Evaluation Tool Alignment of Electropherograms in the Data Evaluation Tool Contents A amp 8V Index Data Handling and Printing c csscssssesccsessesesssesescessseesseseesesseseaeessaeseeasassseatssseeceeeas 137 Organizing Retrieving and Backing Up Data Files 0 cccccscesessessesscsseseessescesesesseesneseeeteeee 137 Saving Data and Assay Files ccccccccessesssssessessesseesesseese
2. Agilent 2100 Bioanalyzer User s Guide for Molecular Assays Edition May 01 Agilent Technologies WARNING For details of safety see the Site Preparation and Safety Manual for the Agilent 2100 Bioanalyzer The Agilent 2100 Bioanalyzer is marked with this symbol when the user should refer to the Site Preparation and Safety Manual in order to protect the Agilent 2100 Bioanalyzer against damage eco 00 CX LabChip and the LabChip logo are registered trademarks of FIADH Caliper Technologies Corp in the U S and other countries Welcome Welcome to the Agilent 2100 Bioanalyzer User s Guide for Molecular Assays This online manual provides novice and advanced users with information needed to successfully run molecular assays with the Agilent 2100 Bioanalyzer A quick look at How to Use This Guide on page 5 explains how easy it is to use this online manual and helps you to get started For Troubleshooting and Maintenance please refer to Maintenance and Troubleshooting Guide Contents A3V Index What s New The major improvements coming with version A 02 10 of the bioanalyzer software are the following e New Assays The bioanalyzer software now supports the improved Protein 200 Plus assay XML Export The bioanalyzer software can now export raw data in XML format optionally including the gel image e Data Organizer As a supplement to the bioanalyzer the Agilent 2100 Bioanalyzer Data Organizer soft
3. Assay DNA 1000 Demo Read 0307 01 13 26 45 Data Path C Programme Agient 2100 Bioanalyzer Bio Modiied 03 07 01 13 30 50 Ladder Peak Mig Time secs Con Area Size BP Conc ng ul Molarity nmol Observations 1 41 75 54 00 15 00 420 424 24 2 4425 31 00 25 00 2 00 12121 3 47 75 32 00 50 00 2 00 60 61 4 53 55 35 00 100 00 2 00 3030 5 60 15 40 00 150 00 2 00 20 20 Gi 6670 4000 200 00 2 00 1515 7 78 55 41 00 300 00 2 00 1010 a 88 05 4200 400 00 2 00 7 58 3 94 55 42 00 500 00 2 00 6 06 T0 704 15 4100 700 00 2 00 433 1 108 35 42 00 850 00 2 00 357 72 110 50 41 00 1000 00 2 00 3 03 13 715 10 42 00 1500 00 210 2 12 Welll PCR Mix 1 25 35 50 53 70 90 100 105 bp Peak Mig Time secs Con Area Size BP Conc Mola Observations 1 41 75 73 72 15 00 4 20 424 24 2 44 25 49 84 2500 530 323 34 ill raz corel ETET ri 127 061 Agilent Technologies 2100 Bioanalyzer Bio Sizing Version A 01 01 N A Click Print to send the combined results to the printer Contents A 65 Vv Index Data Analysis RNA Cy5 Labeled Nucleic Acids d RNA Smear Assays How the Agilent 2100 Bioanalyzer Software Analyzes Data The puropse of RNA assays is to calculate the integrity and concentration of RNA samples Results are calculated after all data for an individual well has been read All RNA assays with the suffix Nano contain
4. Status Ready Type HP DeskJet 895C Where LPT1 Comment HP DeskJet 895C E Pant to file Print range All Pf aR CH Cancel Copies Number of copies 1 When you are finished click OK to close the dialog box Contents A 148 V Index Exporting Data Data can be exported in different formats Export M Result Tables Create a file containing Result Table values I Exclude Markers e Results Tables Well Data T Include Ladder Gel Image T Well Data Exports the signal data as one file per well XML Data I Gellmage Exports the Gel Image as TIFF file ML I Export to XML IV Include Raw Data IV Include Gel Images as encoded jpg r Export Directory C Default C Program Files Data Custom fc Program Files Agilent 2100 Bioanaly el I Create daily subdirectories OK To Export Files 1 Open the file you want to export 2 Click File gt Export The Export dialog box opens Contents A149 V Index 3 Choose an export format Result Tables Create an ASCII text file containing all Result Table values Well Data Export the data in CSV comma separated values format which is suitable for pasting directly into Microsoft Excel or another spreadsheet application Gel Image Exports the Gel Image as a graphic file TIFF format XML Export the data in XML format which is suitable for import of bioanalyzer data files into other software packages This
5. e An error occurred during the saving process If you get this message do the following Contents A 154 V Index Check that the data organizer server PC is up and running From the Windows Start menu select Programs gt Agilent 2100 Bioanalyzer gt Data Organizer gt Connection Wizard and check the settings in the Data Organizer Connection Wizard Uploading Several Chip Data Files To upload several data files 1 From the File menu select Batch Upload to Data Organizer The Batch Upload to Data Organizer dialog box appears 2 In the tree view select the folder that holds the chip data files cld you want to upload Batch Upload to Data Organizer ol x AddSelection 9o celeciA piSelection 3 HemoveSelectedries JX Remove alFies ae Start Batch Upload E Program Files Path H Adobe Agilent 2100 Bioanalyzer Bio Sizing HE Assays CQ Configuration some Bio Sizing exe a Bio Sizingisu B Bio Sizing log gt Cancel eae eet Uplate 0 File s 0 selected File Folder Data A A 155 V Index Contents 3 Click on Add Selection All cld files that were found in the selected folder are added to the file list You can repeat this step with other folders to add more files to the list NOTE To remove files from the list use CTRL click and or SHIFT click or the Select All and Invert Selection buttons to select the files Then click Remove Selected Files To clea
6. 5P Je EF nozul 200 res mo NEB Kb ladder 100 L 2 4 5 E 7 e n 1 ex ia Ready Dif erc Mode hacErpot Awe Pire 7 In this view the gel which is typically shown in small format on the left side of the screen is displayed in the large display and a small single well electropherogram is shown on the left side of the screen One lane of the large gel view will be surrounded by a box This is the selected lane one lane is always selected which corresponds to a selected well in the chip icon the small display will show the well electropherogram corresponding to the selected lane Clicking a different lane will select that lane and the small display will update to show the new well electropherogram Contents A 180 V Index The slider on the right hand side allows you to adjust the brightness and contrast of the gel view When a new run is made the gel display will first be blank and the first lane which corresponds to the ladder well will be selected As data is acquired in the first and subsequent lanes wells containing samples the selection box will increment to show the well and lane that is currently acquiring data If you select a lane well that is earlier in sequence than the current well however the display will no longer increment as new data is acquired but will remain on the selected lane well Moving the mouse pointer over a gel in the l
7. Contents A 161 V7 Index NOTE Positioning the cursor over a tool on the tool bar or over other items in the workspace and leaving it there momentarily will cause a Tool Tip to appear listing the name of the tool Often this will be enough to describe the item s function Printing Help You can print specific help topics or print entire sections of online Help To print an entire section from the Contents page click the Print button that appears along the bottom right side of the window To print a single help topic go to that topic and click the Print button that appears at the top of the window Contents A 162 V Index Types of Help Available Three types of help are available within the Agilent 2100 bioanalyzer software General Help By clicking the Help menu and choosing Contents and Index you can view the Help topics that are grouped by subject matter into books or search for a keyword and jump to a topic Context Sensitive Help Pressing F1 will provide help that is specific to the active window Hotspots and Links Certain items in the help files are links to other types of help Clicking items that are underlined or over which the cursor changes to a pointing hand will cause additional help to pop up or will take you to another help screen containing more information For more information see Using the Help Function Contents A 163 V Index Mouse Notation Conventions in Help When you see t
8. Molarity where molarity is measured in nanomoles per liter nmol L concentration is measured in nanograms per microliter ng L size is measured in base pairs bp 660 is the molecular weight of one basepair Additional information about the peak such as possible comigration or expected fragment indication Contents A61V Index Reanalyzing a Data File Occasionally you may wish to open and view or reanalyze a data file that was run and saved previously The raw data values are saved in the data file along with the analysis settings that were chosen for the run so that the data can be reanalyzed with different settings To do this 1 Click File gt Open 2 Choose the filename from the list of data files 3 Click OK If you have no unsaved data currently open the chosen file will open allowing you to view edit the results If you have unsaved data open a dialog box will ask if you want to save the current data first The items that can be changed for reanalysis are Global peak find settings e Individual sample peak find settings chosen in the sample information pane to the right of the Results Table in the single well view window see Settings Tab on page 178 e Expected base pair size for certain assays Gel color Sample names and comments e Exclude peaks from analysis e Reassign upper lower markers Alignment or no alignment with ladder peaks Contents A 62 V Index e Use of ladder r
9. Save As OK Cancel Contents A 108 V Index Assigning Upper and Lower Marker Peaks For each sample the upper and lower marker peaks are assigned first and then the data is aligned so that the well markers match the ladder markers in time This allows the size determination of the sample peaks The first peak above a certain threshold is assigned to be the lower marker and is then offset to match the lower marker in the ladder The upper marker is then assigned either to the last peak in the sample well or to the peak nearest to the ladder s upper marker See Aligning or Unaligning the Marker Peaks 111 for an example of assigned marker peaks If you see unexpected peaks in the ladder analysis or the markers are set incorrectly you can exclude peaks manually from the ladder NOTE Excluding a peak or manually setting a peak to be an upper or lower marker may cause errors in analysis You can move the boundary between the Results Table and the well graph up or down to increase or reduce the amount of space allotted to the Results Table making it possible to see all of the results at once Contents A 109 V Index Right clicking in the result table of a adder well causes this pop up menu to appear Right clicking in the result table of a sample well causes this pop up menu to appear Aligning or Unaligning the Marker Peaks The upper and lower markers are then aligned to the ladder markers by re sampling th
10. Run sample m to p I Edit samples after start This dialog box appears when you click the Start button above the chip icon You can enter a file prefix other than the one shown in the dialog box When you are ready to begin the run click Start Contents A196 V Index General Chip Information Dialog Box This dialog box appears also if you select File gt Properties The General Chip Information dialog box consists of several tabs which you can use to enter study information and sample settings Two other tabs show you information on the data file and a chip run summary If the Data Organizer Client is installed on your system an additional Custom DO Information tab is available Use the Export button to export the data in CSV comma separated values format which is suitable for pasting directly into Microsoft Excel or another spreadsheet application Use the Import button to import previously exported existing data on the samples NOTE The format needs to be the same as in the dialog box Use the Run Log button to display the Run Log dialog box which displays information about the particular run including the date and time of the run any problems that occur the assay used o generate the data and the name for the saved data file File Information Tab The File Information tab is part of the General Chip Information dialog box The tab shows you information on the data file that is automatically created by t
11. This file contains chip and sample attributes in a structured format supporting easy data exchange Report file pdf This file contains the assay summary gel like view electropherogram and results tables It can be printed on any printer Description file env This file contains information about the upload computer upload path file date and file name NOTE The names of the export report and description files are derived from the name of the cld file Example xml Example pdf and Example env would be generated and uploaded when you upload Example cld Contents A 153 V Index Uploading the Currently Loaded Chip Data To upload currently loaded chip data 1 Open a data file cld or run a new assay 2 If any setting for example peak find settings has been changed or information e g custom DO Information added after the end of the run save the file Otherwise the changes will not come to effect in the Data Organizer 3 From the File menu select Upload to Data Organizer The following message appears Upload file BioSizing_DNA 7500_00000_2001 09 05_17 27 27 cld to Data Organizer 4 Click OK to confirm When the upload is finished you will get one of the following messages The data was successfully saved in the database The existing data in the database was successfully updated Database Rebuild is running no upload possible until rebuild finishes
12. 3 Click Apply Contents A49V Index 5 10pgdT24ori Markdr Y 8 Fluorescence Start Time total area Region Upper i Marker End Time 5 w 65 7 w so 35 o0 o5 Time seconds 7 The results related to the defined region are displayed in the Results table If you have defined several regions you can select the desired region in the Results table and edit them on the Regions tab From Lower limit of the region in bp To Upper limit of the region in bp Corr Area The area under the peak within the region of total Area Observation Percent of the total area that is defined by the start and end time markers Additional information about the peak Contents A50V Index Global regions that are valid for all wells can be defined by choosing Assay gt Assay Properties and selecting the Regions tab You can enter the lower and upper limit of the range and the color of the range f Demo DNA 7500 Smear Properties xi Summary Ladder Analysis Global Peak Find From bp To bp 1 foo B TALL HA a Contents A51V Index Changing the View of the Results A number of different options are available for viewing the data after it has been acquired by the Agilent 2100 bioanalyzer None of these options change the raw data but rather provide different means of viewing the results Overlaying Electropherogr
13. Copy Gel Copy Single Line Copy Graph Copy Results Table Choosing any of these copy commands places a copy of the chosen item to the Windows clipboard You may then paste the item into a word processing graphics or other application Choosing Copy Gel from the Edit menu always copies a large gel picture such as would be seen if you viewed a large gel display with the lane labels as part of the graphic Choosing Copy Single Line from the Edit menu copies the selected line of the large gel picture as bitmap into the clipboard The size of the image that is placed on the clipboard when copying a graph depends on the display mode at the time you choose Edit gt Copy Graph or right click the mouse and choose the Copy Graph command from the pop up menu If you right click and choose to copy while the cursor is over the large well graph or if you choose Copy Graph from the Edit menu while viewing a single well display a large sized graphic the same size as that shown in the large display of the well graph will be placed in the clipboard Contents A 159 V Index Copying the result table either by choosing this command from the Edit menu or by right clicking in the result table area and choosing the copy command from the pop up menu causes ASCII information to be placed in the Clipboard The following text is an example of a DNA result table data that was copied from a sample run Bio Sizing_00589_2000 05
14. Lower marter Upper marker Observations Upper Mate Observations Lawer Maer Upper Mae Demo DNA 7500 A 01 20 1 211 Page 1 Tanay Deine DNA 7500 Fond 275001 119645 AM AD 20 SITT Daa Pan Z1SyatersiDemo FlesDere AGT 20_Si211 Malie 24901 144308 Ald A120 SZH Agen Technologies 2100 Bionnatyzer Bio Sizing Version A101 NIA Prine 471001 3141 20 PM 4145Y Index Printing 4 wells per page Electropherogram Demo DNA 7500 A 01 20 SI 211 Contents A 146 Y Index Setting Up Your Printer If your printer is not functioning correctly you must select and properly configure the appropriate printer driver Consult the printer manufacturer s instructions or the Windows documentation to find out how best to set up the printer driver From the File menu choose Page Setup Page Setup 21 x m Paper size z Source AutoSelect Tray z Orientation Margins inches Portrait Left fos Right fos C Landscape Top 0 25 Bottom o 25 Cancel Biner Make selections that are appropriate for your particular printer Clicking the arrows to the right of selection boxes will provide you with different options Contents A147 V7 Index Clicking the OK button in the Print menu opens another dialog box allowing you to select options for the default printer or to choose a different printer Print Properties Printer Name
15. 06_ 09 19 49 Sample 1 Peak Mig Time secs Area Size BP Conc ng ul Molarity nmol l 1 30 30 2 57 50 40 121 21 2 32 80 3 24 100 7 5 113 85 3 63 60 2 96 3000 4 0 2 02 The first line shows the name of the saved data file followed by the name of the sample from which the result table data came The second line provides the headers for the rest of the information which includes the peak number the migration time in seconds the peak area the size of the peaks in base pairs the concentration in nanograms per microliter and the molarity in nanomoles Contents A 160 V Index Using Help The Help system enables you to retrieve the information you need quickly and then return to your work Help appears in a separate window on your screen For quick access you may keep the Help window displayed on top of or behind the application You can also print specific topics from the online Help system Context sensitive help is also available Contents and Index When you click Contents and Index from the Help menu the Help window opens allowing you to do one of the following things Click the Contents tab to display conceptual and how to information Click Index to search by a name or concept Context Sensitive Help The context sensitive Help displays information that is relevant to the current window or dialog box displayed by the Agilent 2100 bioanalyzer software To access context sensitive help click the Help button or press F1
16. 13 Agilent 2100 bioanalyzer software 20 Agilent 2100 bioanalyzer handling 28 alert 208 alignment automatic 133 manual 134 alignment principles 133 analysis time window 78 turn off on 194 Contents Analysis On Off icon 169 area results table 61 assay information 173 menu 190 properties 192 assay files saving 138 assay properties 37 assay properties dialog box 173 211 auto export 210 auto print 210 automatic alignment 133 B background fluorescence 58 backing up data files 137 baseline correction DNA 59 drift 38 lokal peak 38 parameter for peak finding 214 plateau 40 zero 209 baseline plateau 214 A234 V7 Index baseline subtraction 136 baseline force to zero 58 batch upload to data organizer 152 Bio Sizing launcher 21 brightness 56 browser 244 bubbles 25 bundles 231 c calculation RNA concentration 68 calibration protein assays 113 calibration curve DNA 35 calibration procedure 35 capillary electrophoresis 230 cell lysates 192 change your data analysis 69 101 changing to gel view 88 channels 228 check box 164 checking hardware 194 chip 13 temperature 194 chip data file 153 chip icon 166 171 172 chip inserted 172 chip receptacle 14 Contents chip run summary 200 201 clean electrodes 28 column fractions 192 combining results DNA 63 Proteins 125 RNA 93 combining results table 216 communication problem 171 compare results 194 computer 227 concentration 35 RNA 68 C
17. 2100 bioanalyzer the data organizer software G2945AA provides central management and security of bioanalyzer data and allows you to share data files with other scientists who have been granted access to the data organizer server Uploading chip data files to the data organizer server is possible directly from within the bio sizing application After successful installation of the data organizer client refer to the Data Organizer Installation Guide for details two new items are added to the File menu of the bio sizing application Upload to Data Organizer Uploads only the currently loaded chip data Batch Upload to Data Organizer Uploads a group of chip data files This command opens a dialog box where you can select the cld files to be uploaded The following figure shows the modified menu File Open CtreO ed Save Save As Save Selected Wells As x Export Upload to Data Organizer Batch Upload to Data Organizer amp Print Ctrl P CiS Contents A 152 V Index NOTE The 2100 bioanalyzer data organizer version A 01 01 supports the management of files generated by the 2100 bioanalyzer system software for molecular assays bio sizing Organization of data files from the 2100 bioanalyzer system software for cell fluorescence assays is planned for the next release of the data organizer software For each cld file you upload actually a file set is uploaded Chip data file cld Export file xml
18. Curve Gone i Green on Black Magenta on White E Red on Black E Pseudo on black By Chip Run Summary Contents A89V Index Force Baseline to Zero Since all electropherograms show some amount of background fluorescence the bioanalyzer software automatically sets the baseline to zero fluorescence units To remove the zeroing select Tools gt Options gt Advanced and disable the Zero Baseline box Ladder Zero Baseline 20 Fluorescence 1 t 16 a 2 a 3 a 5 ss 61 os n 75 s Time seconds T Ladder Non Zero Baseline asf Fluorescence at Contents A 90 V Index The Results Table The Results Table appears below the single well view in the large display area This table provides the following information Fragment Number The order in which the fragments were detected Fragment Name A user assigned or predefined name for the found fragment Typically 168 238 for Prokaryote assays or 18S 28S for Eukaryote assays Start Time secs Shows the start time for the peak The start and end times are also represented on the electropherogram by diamond shaped points on the peak baseline in the same color as that shown in the RNA tab Dragging a diamond will change the start or end time and alter the baseline drawn between the diamond markers End Time secs Shows the end time for the peak The start and end times are also repr
19. Opens the Calibration Curve dialog box allowing you to view the linear regression line for calculating the concentration of proteins in relation to a standard protein Contents A 188 V Index Graph Menu Graph Scale to Selected Scale All No Peak Info Show Data Points Cto ESS STs Ba Copy Graph Ctrl C Peak Time Peak Height v Peak Area Peak Size Peak Concentration Peak Molarity Scale to Selected Scale All Undo Zoom Undo Zoom Completely Peak Info Show Data Points Scales the data in all wells to the data in the selected well Scales display of each well to itself allowing all of the peaks to be visible Holding down the Shift key and choosing Scale All causes all of the wells to be scaled relative to each other Undoes zoom step by step Double clicking in the single well display performs the same function Returns to the standard unzoomed view of the single well Allows you to choose the type of information that is shown in the Result Table of the single well display Default is Peak Number Enables disables the display of the data points used to generate the graph Data points are visible only in the single well display Contents A 189 V Index Assay Menu Assay dsDNA BNA Protein Other Smear Demos C Demo DNA 7500 Properties Save Assay As ywrvrVwYY Start Contents DNA 1000 DNA 12000 DNA 500 DNA 7500 Eukaryote To
20. Peak Find within this dialog box allows you to change the peak find settings Contents A173 V Index Large Display Large Multiwell Graph View This area of the workspace shows a multiwell view default of the data received from the chip a single well display of a selected well or a gel view When the multiwell or single well views are selected the small display area shows the gel view and vice versa Agilent Technologies 2100 Bioanalyzer Bio Sizing Bic DNA 12000_00000_2001 07 05_16 09 32 Hie Edt View Graph Astay Took Heb 0 64 008 88 8 a raae 7 Data File BioSizing_DHA 12000_00000_2001 07 05 16 08 32 7 E Diu eat 05 070 f 0832 Assay Demo DNA 12000 nal L 1l EANN NEE Ti ir ADI neha i A ANAA N wey i A Ad2 Pvul High Mass Ladder Synthetic DNA Ladder NEB 1hb ladder Adz Dral H Lo i MW a uad Pe A Aigi H Ad2 P vu High Mass Ladder ire MEN J L pl Be lu U Synthetic DNA Ladder Fead fine Demo Mode Contents A 1740F AaoEssor auo P 5 Index This is the default view when you start the Agilent 2100 Bioanalyzer software As data is acquired the selected well Sample 1 in the example above will increment to the well that is currently being run and the data will appear in the display in real time The screen above shows the large display after
21. a 50bp DNA fragment as lower marker This double stranded fragment runs in the posi ion of a 25 nucleotide RNA transcript The data analysis process for RNA and the Cy5 labeled nucleic acids assays consists of the following steps 1 Raw data is read and stored by the system for all the individual wells 2 A software algorithm filters the data and plots the resulting electropherograms of all wells You can change the settings of the filtering algorithm after the run and reanalyze your data 3 Fragments are identified for all wells and tabulated by migration time You can change the settings of the peak find algorithm after the run has finished and reanalyze your data Contents A 66 V Index 4 An RNA ladder a mixture of 6 RNA transcripts of a well defined size and total concentration is run first from the ladder well see the electropherograms below The ladder information is preset in the assay and can not be changed Ladder Fluorescence Time seconds Sample RNA Nano ladder total RNA Contents A67V Index 5 For the Eukaryote or Prokaryote Total RNA assay two time windows that are determined dynamically based on the ladder run are used to assist in detecting the ribosomal RNA bands either 18S and 28S for eukaryotic RNA or 16S and 23S for prokaryotic RNA These windows are delineated by short dashed lines shown in the same color as the fragment designator actual
22. been altered in any way Choosing this command while using one of the default program assays will cause an error message to appear since those assay files are read only and will open the Save As dialog box instead Opens the Save As dialog box allowing you to save the assay as it is currently configured under a different assay name Opens the Start dialog box allowing you to enter information regarding the run Clicking Start in this dialog box will initiate the run Note that the Start dialog box also allows you to change the file prefix that will be used in automatically naming the data file Contents A 193 V Index Tools Menu Tools Tum Dif Analysis Ctra p3 ent Temp e Montar View Log File Options Turn Off On Analysis Diagnose Instrument Compare Results Temperature Monitor Contents The default is analysis on which causes the marker peaks run with the samples to be aligned to marker peaks in the ladder Choosing this command turns analysis off which removes the marker peak alignment Opens the Diagnose Interface for checking the hardware components of the Agilent 2100 Bioanalyzer Opens the Data Evaluation program as a stand alone program You can compare results from wells within a single run or between runs or assays that have been saved previously Displays actual chip baseplate temperature A194 V7 Index View Log File Four types of log files are maint
23. function creates one XML file per data file containing information about the used assay and the obtained results Optionally you can add the measured data points x and y coordinats for the electropherogram and the gel like image as encoded jpeg 4 Choose an Export directory 5 Clicking the OK button in the Export dialog box causes the Export Data dialog box to appear Accept the default filename shown in this dialog box or enter a different one The file extension will automatically be csv for well data txt for result tables tif for Gel images and xml for the XML format Accept the default location for saving this file or choose a different location if desired 6 Click OK and the data will be exported to that filename in that location Contents A 150 V Index Auto Export It is possible to export every data file generated automatically via Auto Export 1 Click Tools gt Options The Options dialog box opens 2 Choose the Advanced tab 3 Enable the Auto Export check box 4 Click the Settings button to open the Auto Export dialog box 5 The Auto Export dialog box gives you exactly the same choice as the Export dialog box Choose an export format see page 149 6 Choose an Export directory If you have a large output of data files it is advisable to activate the Create Daily Subdirectories check box Contents A 1517 Index Uploading Chip Data to the Data Organizer As an extension to the
24. individual wells You can use this information later on when searching for data in the data organizer repository For information on how to configure the Sample and Chip Custom Fields please refer to the Data Organizer Users Guide Contents A 200 V Index Chip Run Summary Tab The Chip Run Summary tab is part of the General Chip Information dialog box and is displayed when an assay is finished if General Chip Information x File Information Study Information Samples Information Chip Run Summary vA Status Assay completed Read 05 07 01 16 09 32 Sample Observation vi 3 sample peak s found in NEB 1 kb ladder 14 sample peaks found in Ad2 Dra l 10 sample peakfs found in Ad2 Bgl I 7 sample peak s found in Ad2 Pvu E sample peak s found in High Mass Ladder 12 sample peak s found in Synthetic DNA Ladder 3 sample peak s found in NEB 1kb ladder 13 sample peak s found in Ad2 Dra l Possibly no restriction digest 7 sample peak s found in Ad2 Pvu 6 sample peaks found in High Mass Ladder 12 sample peak s found in Synthetic DNA Ladder SS8ES855888 Bodgeor H20Nn Help spot Fun Log ok Cancel It shows a list of the samples been measured and error messages if the measurement was incorrect Contents A201 V7 Index Open Data File Dialog Box Lookin E Data z E S a BioSizing DNA 1000_00000_2001 10 01_14 32 01 cld Filename f Files of typ
25. information areas are located just above the large display and show the following information Data File BioSizing_MRHA Hano_00000_2002 02 22_12 45 18 Read 02 22 02 12 45 18 Contents A172 V Index Left side of the display file information The name of the data file if saved otherwise the area on the left is blank The date and time the data file was created Assay Demo mRNA Nano Right side of the display assay information The assay that was used to generate the data on the right Should an error occur with data collection during an assay a red circle with an X in it will appear to the left of the file information and a third line of data file information will appear listing the type of error that occurred Clicking on the red circle with an X in it will open the help for that particular error message allowing you to view possible causes for the problem as well as any potential solutions Data File BioSizing_mRNA Nano_00000_2002 02 22_ 12 45 18 Read 02 22 02 12 45 18 Status Aborted by the user You can add or amend notes for the run by accessing the notes section of the File Properties dialog box double click the filename shown above the large display The File Properties dialog box that appears also contains a button allowing you to view the Run Log for that data file Double clicking the assay information shown on the right side above the large display opens the Assay Properties dialog box The tab labeled Global
26. inverse of this value is used to determine the peak end Determines the minimum amount of time that must have elapsed after hreshold was exceeded A parameter that assists in finding peaks The signal is recognized to be at baseline whenever the slope of the data is less than the Slope Threshold setting either positive or negative for longer than the time set for the Baseline Plateau This setting rejects brief low slope areas such as those found between non baseline resolved peaks Default enabled This setting causes the bioanalyzer software to use he values defined by the assay for ladder data instead of data obtained rom the ladder run with the assay Default enabled Baseline flattening mechanism corrects for drifts in baseline If analyzing cell lysates or crude extracts it might be required o turn the baseline correction off Default disabled If checked the calibrated concentration will be shown for all sample proteins calculated based on the calibration curve Otherwise the calibrated concentration will only be shown for the protein corresponding to the calibration protein Contents A105 V Index You can change all peak find settings except the Baseline Plateau for individual wells In the lower right pane of the single well display to the right of the Results Table are four tabs The Settings tab shows the peak find settings that are currently in effect for that well Changing the settings shown on this
27. nm Ss Ded a 30 35 40 45 Time seconds NOTE The software performs alignment by default Turning alignment off suspends data evaluation until you turn it on again Contents A99 V Index 7 The standard curve in conjunction with the markers is used to calculate protein sizes for each well from the migration times measured 8 To calculate the relative concentration of the individual proteins in all sample wells the area of the upper marker with known concentration is compared to the area of the individual sample proteins NOTE The software allows you to define upper and lower markers yourself However a change in the selection of the upper markers will lead to quantitative changes of the calibration procedure and will therefore alter the entire data evaluation Contents A 100 V Index Changing Your Data Analysis Protein Changing the Settings of the Data Evaluation Algorithm Different sets of parameters can be changed in the software in order to modify the data evaluation for sample analysis Filtering parameters Peak find parameters for all wells peak height for individual wells Time window for analysis Assigning upper and lower marker peaks lt Aligning or unaligning marker peaks e Using calibration Changes can be made before a new run is started or to reanalyze the data from a previous run Filtering Parameters The first step the software takes in analyzing the data is to apply data filtering H
28. on View gt View Gel The main window will change and display the results in a format as it would be generated by a slab gel device Ladder NEB1kb Ad2 Dral Ad2 Bglll Ad2 Pvul High Mass Synthetic NEB 1kb Ad2 Dral Ad2 Bglll Ad2 Pvul High Mass Synthetic ladder Ladder DNA ladder Ladder DNA 17000 e o ee eS o a D 10380 7000 a z ee E SES SSE G _ 1 5000 4 a 3000 Posrsap es 2000 ng ul 4 79 nmol 1000 700 500 T 300 100 50 n a ee Se A SS ces ees Sees ems SD T 1 2 5 6 7 0 n t Contents A55V Index Moving the mouse pointer over a gel in the large display will cause numbers to appear next to a crosshair pointer With a DNA assay you will see the base pair measurements for the area of the lane beneath the crosshair of the pointer shown by a If the cursor is positioned over a recognized band the cursor will change to show a target and the concentration and molarity will also be shown The slider on the right hand side allows you to adjust the brightness Contents A56V Index Different gel display colors are available by choosing View gt Gel Color and then choosing one of the color schemes from the drop down menu The colors are designed to approximate actual gel staining and imaging techniques Blue on white for example simulates a Coomassie gel often used with proteins The Pseudo colo
29. printout with all wells For printouts of Electropherogram or Combined Result Tables you can select individual wells to be printed The Options field gives you additional options on how to arrange the printouts Include Ladder can be selected or deselected if All wells is selected Contents A140 VY Index Clicking OK in this dialog box causes another printer specific dialog box to appear allowing you to set the number of copies you would like to print along with other settings that determine the appearance of the printed document For more information see Setting Up Your Printer on page 147 Contents Al41V Index Printing an Assay Summary Report Contents A 1427 Index Printing a Gel Report CL75_10_00022_1999 06 10_16 35 36 Read 06 10 99 04 35 pM page 2 Data path cx Filesi ro ser Bio sizi a Daga path c program Eilgs uP 2100 Bio analyzer eio sizing oat Printing an Electropherogram Demo DNA 7500 A 01 20 1 211 Pags 1 Demo ONA 7500 p ihe 201801 144308 AM A120 SZH Daa Pam _Z3SyatenDemo FiesDamo A 01 20 5121 Sample 6 450 Time seconds Pesk MigTime Ams Sue Cone Molarity Observations aeea BP oglu amti R wu 2 25151 Lower Markee 2 sre ons mooo z 3 i205 1705 tw Be 170 5 tem are 25 28 Jeo H va mo ma 27 15 7 dars saor 330 150 s f
30. tab will affect this well only to change the settings that affect all wells click the Global button to open the Assay Properties dialog box and then click the Global Peak Find tab Sample Settings Results Enors Min Peak Height 20 Global Min Peak Width 02 Reset Slope Threshold 40 I Baseline Correction If you change the Global peak find settings after making individual well setting changes the following prompt will appear 7 Do you want to override custom well settings te ca Contents A 106 V Index Choosing Yes causes any changes made to the peak find settings for individual wells to be discarded and applies the global peak find settings to all wells Choosing No allows individual wells to retain changed peak find settings NOTE This prompt appears whenever at least one of the samples has different local settings Contents A107 V Index Time Window for Analysis The Start Time and End Time parameters on the Global Peak Find tab see figure below define the time window within peaks will be found Demo Protein 200 Plus Properties xi Summary Ladder Analysis Min Peak Height 2 Min Peak Width Secs 0 2 Reset Slope Threshold Sec 4 Start Time Secs 15 End Time Secs 47 Filter Width Secs 0 5 Baseline Plateau Secs 0 3 Polynomial Order 6 I Calibrate all Proteins M Baseline Correction IV Exclude Ladder keep current values
31. the pipette at the edge of the well may lead to bubbles and poor results Holding the pipette at a slight angle will ensure propper dispensing of the liquid e Use a new syringe and cleaning chip with each new LabChip Kit Contents A25V Index Reagents and Reagent Mixes General e Handle and store all reagents according to the instructions given in the specific Reagent Kit Guide Keep all reagents and reagent mixes for example the gel dye mixture refrigerated at 4 C when not in use for more than 1 hour Reagents might decompose leading to poor measurement results Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use Protect dye and dye mixtures from light Remove light covers only when pipetting Dye decomposes when exposed to light Gel and Gel Dye Use gel dye mixture within four weeks of preparation or as specified in the appropriate Reagent it Guide The gel dye mixture might decompose and lead to poor measurement results Samples Refer to the assay specific Reagent Kit Guides for maximum allowed sample and salt concentration For proteins Use 0 5 ml tubes to denature samples Using larger tubes may lead to poor results caused by evaporation Contents A26V Index Chips Prepare and run chips within 10 minutes Longer chip preparation times may lead to evaporation of buffers and to bad chip performance Vortex chips for appropriate 1 minute not required for p
32. the large display View Gel Shows the gel in the large display Combined Results Opens the Combined Results View dialog box to set the options used to display the combined results tables Previous Well In any display decrements the view to the previous well or lane If you are viewing Sample 1 clicking this button or pressing the left arrow button on the keyboard takes you to the ladder well lane If you are viewing the ladder well clicking this button or pressing the left arrow button on the keyboard takes you to the last well lane Contents A 187 Y Index Next Well Gel Color Standard Curve Calibration Curve In any display increments the view to the next well or lane If you are viewing the ladder clicking this button or pressing the right arrow button on the keyboard takes you to Sample 1 If you are viewing the last sample clicking this button or pressing the right arrow button on the keyboard takes you to the ladder well lane This pull down menu presents different color choices for viewing a gel in the large display The colors are designed to approximate various actual gel staining and imaging techniques The Pseudo color choice provides more detail 1280 colors since it maps the signal into a larger color space than is available with the other monochrome options 256 levels of brightness Opens the Standard Curve dialog box allowing you to view the DNA ladder as a curve with a point to point fit
33. used as lower or upper markers Default enabled in all RNA nano assays disabled in all other RNA assays This setting causes the bioanalyzer software to subtract an additionally added lower marker If you want to compare assays without a lower marker with assays containing a lower marker the lower marker s area has to be subtracted to get a corresponding total area Contents Anv Index You can change all peak find settings except the Baseline Plateau for individual wells In the lower right pane of the single well display to the right of the Results Table are four tabs The Settings tab shows the peak find settings that are currently in effect for that well Changing the settings shown on this tab will affect this well only to change the settings that affect all wells click the Global button to open the Assay Properties dialog box and then click the Global Peak Find tab Sample Settings RNA Results Enors Min Peak Height 05 Global Min Peak Width 05 Reset Slope Threshold 0 8 Baseline Start Time 22 00 Secs End Time 69 00 Secs IV RNA alignment Cancel Apply Contents ATAV Index If you change the Global peak find settings after making individual well setting changes the following prompt will appear 7 Do you want to override custom well settings te coal Choosing Yes causes any changes made to the peak find settings for individual wells to be discarded and applie
34. used to define the power series applied to fit the raw data The higher the number you set the more the fit function will follow the noisy raw data curve As a result the noise level of the filtered curve will increase Filter Width defines the data window in seconds used for averaging The broader the filter width the more raw data points are used for averaging As a result the noise level will decrease but peaks will become lower and broader Overall changing the Filter Width has more effect on the result of the filtering procedure that is applied than does changing the Polynomial Order Peak Find Parameters After data filtering the Peak Find algorithm locates the peaks and calculates the local peak baselines The algorithm begins by finding all the peaks above the noise threshold in order to determine the baseline after which any peaks below the noise threshold are rejected A local baseline is calculated for each peak to allow for baseline drift Contents Aay Index The four peak find parameters that can be changed Min Peak Height Slope Threshold Min Peak Width and Baseline Plateau are shown below Choosing OK sets the parameters for all the wells X Eukaryote Total RNA Nano Properties x Summary Ladder Analysis Min Peak Height 0 5 Min Peak Width Secs 0 5 Reset Slope Threshold Sec 0 8 Start Time Secs 20 End Time Secs 69 Filter Width Secs 1 Base
35. with labels Fragment start end times additional peaks or delete peak Alignment or no alignment with ladder peaks Contents A92V Index e Use of ladder run with samples or use of internal assay ladder Assay you can save the changed settings under a new assay name if desired NOTE If you save the data file after making changes it will keep a record of the assay in use if a new assay name has been saved it will use the settings from this assay the next time the file is opened gel color well names and peak find settings that were in effect at the time the file is resaved If you don t want to change the original file choose Save As and give the file a new name or save it to a different location Combining Results If you want to combine the results of different wells you can select the wells with the results to be combined You then can print a table view of the results To do this 1 Click View gt Combined Results 2 Select the wells to be combined 3 Click OK to display the combined results in a table view The items that can be changed for combining results Well range All Wells to combine the results of all measured wells e Wells to combine the results of selected wells Contents A93V Index Options Exclude Markers to display the values without the markers Include Ladder to display the values of the ladder in a separate table Combined Result View Contents A
36. 0 4 30 4 20 10 4 trp 8 A T Lola TT 08 amp 0438 Pa 6 EK 5458 Hai amp ofar 25 30 35 40 5 50 55 60 90 Time seconds T Sample Settings Results Enors Min Peak Height 80 Global Min Peak Width E epee Slope Threshold os I Baseline Correction Cancel Boal Contents A32V Index 5 Astandard curve of migration time against DNA size is plotted by using linear interpolation The standard curve derived from the data of the ladder well should resemble the one shown below i Standard Curve ox Point to Point Fit G Cuve Fil Lader Fremeni E g 5 E Contents A33V Index 6 Two DNA fragments are run with each of the samples bracketing the DNA sizing analysis Called lower and upper markers these are internal standards and are used to align the ladder analysis with the individual sample analysis The figure below shows an example of assigned marker peaks in a sample well 400 4 350 4 300 250 200 150 4 Fluorescence 100 50 4 Lower Marker Upper Marker 1 motte ttt oom bo N NOTE Contents T T T T T 30 35 40 45 50 55 60 65 Time seconds yo y y h yo Th I 7 0 76 80 85 90 The software performs the alignment automatically You can turn off the alignment at the end of a run however no automatic data evaluation will occur until the alignm
37. 00 ng rat besin mRNA tT Start Timel i End Time Fluorescence Baseline Time seconds Move the cursor over the left long dashed line lower baseline set point and drag the line to the desired position Do the same with the right long dashed line upper baseline set point until the baseline is flat NOTE Changing the baseline set point will change the calculated RNA concentration Contents A719 V Index Aligning or Unaligning the RNA Peaks Nano Assays The signals are aligned to the lower or upper marker that you have manually set In the example below the lower marker is manually set to the first fragment You can align the signals also to the upper marker which must be manually set All samples are aligned to the master sample red lines The master contains the manually set markers If the lower marker is set automatically no marker manually set the first sample containing a lower marker is selected as master L T Data before alignment Alignment to the upper and lower marker Contents A80V Index On the Settings tab you can enable RNA alignment for each well separately Select the RNA alignment check box and click Apply to align the signals of the selected well Clicking Global opens the Assay Properties dialog box Select RNA Alignment and overwrite the current assay settings to align the peaks of all wells Sample Settings RNA Res
38. 000 1195 2058 651 818 1039 2058 A SSA f ss5s95959595g5 F 651 818 1039 Well 12 Synthetic DNA Ladde 175 252 395 523 4 Chip Comments Reset Peal al ok Cancel Use the Sample Information tab to change the sample name to include comments and expected fragments and the fragments range and the restriction digest for DNA sizing only Enter any notes about the run in the chip comments box at the bottom When you perform a protein assay you can enable the use of calibration and enter a calibration value Use the Reset button to undo the changes you made Use the Apply to All button to apply the settings of one row to all rows of the column Contents A 199 V Index The Sample Information tab can also be accessed through the sample tab in the Single Well View See Large Single Well Graph View on page 176 Custom DO Information Tab The Custom DO Information tab is part of the General Chip Information dialog box and is only available if the Data Organizer Client is installed i General Chip Information x File Information Study Information Sample Information Custom DO Information Chip Run Summary Chip Specific Chip Custom Field 1 lip Custom Field 2 Chip Custom Field 3 Chip Custom Field 4 Sample Specific Reset Doster It provides a number of input fields where you can enter custom information about both the chip and the
39. 2 01 6 25 60 8 56 44 00 1110 353 f p3 32 00 30 34 0 00 so zolic 17 Agilent Technologies 2100 Bioanalyzer Bio Sizing Version 4 01 01 N A This dialog box appears when you have chosen your options in the Combined Results View dialog box and you click OK The results are different for DNA RNA and protein measurements When you click Print the data is sent to the printer You also can print combined result tables using the Print dialog box see Printing a Report on page 140 Contents A211V Index Print Dialog Box gt Print M Assay Summary I GelLike I Electropherogram I Combined Result Tables r Well Range Options Alliwele J diwe Wels Te Thelade ladder Enter well number and or well ranges separated by commas Example 1 2 5 12 L for Ladder ez When you choose Print from the File menu the dialog box shown above appears providing four options for printing You can choose from one to all four options Assay Summary and Gel Like always produce a printout with all wells For printouts of Electropherogram or Combined Result Tables you can select individual wells to be printed The Options field gives you additional options for these choices Clicking OK sends the print request See Printing a Report on page 140 for more information Contents A218 Y Index Save Data As Dialog Box Save in fa Data x ce a BioSizing_
40. 221 title bar 166 167 tool bar 166 168 tools 25 Contents tools menu 194 U unzoom tool 169 upload messages 154 upload to data organizer 152 Upload to Data Organizer menu com mand 152 uploading chip data 152 upper and lower marker definition 100 upper and lower marker peaks 44 upper and lower markers defining 35 upper marker 44 URL 25 view menu 187 WwW web site 25 well data exporting 149 well graph view large 176 well graphs 52 A 241V Index X XML data exporting 149 file 153 Z zero baseline 209 zeroing baseline 58 zoom 189 About This Guide Copyright 2001 2002 Agilent Technologies Use Reproduction and Distribution is subject to approval of Agilent Technologies Edition 02 02 p n G2941 90002 Adobe and Acrobat are U S registered trademarks of Adobe Systems Incorporated Microsoft Windows and Windows NT are U S registered trademarks of Microsoft Corporation LabChip and the LabChip logo are registered trademarks of Caliper Technologies Corp in the U S and other countries RNAseZAP is a registered trademark of Ambion Inc Contents A243 V7 Index Did You Know The Agilent Technologies logo on the cover page launches your PC s default browser and goes to the lab on a chip pages Try it here Agilent Technologies You link to the separate Maintenance and Troubleshooting Guide by clicking on the cross references Contents A247
41. 4 4 22 45 1727 21 23 22 30 7 12 5 23 55 53 38 23 00 69 10 22 01 g 25 60 856 44 00 1110 353 za 3034 To Ea zalica Agilent Technologies 2100 Bioanalyzer Bio Sizing Version A 01 01 N A OK Click Print to send the combined results to the selected printer Contents A127 V Index Data Evaluation The Agilent 2100 bioanalyzer software allows you to overlay and visually compare two or more electropherograms from the same chip However it is not possible to adjust these graphs in any way nor can you compare electropherograms from different runs The data evaluation tool extends the capability of the Agilent 2100 bioanalyzer software by allowing you to compare up to 12 electropherograms recorded by the Agilent 2100 bioanalyzer These can be from the same or different runs You can also adjust the alignment of the curves through either automatic or manual settings and you can view the graphs in different ways to enhance the presentation of the data Contents A 128 V Index Starting the Data Evaluation Tool The data evaluation tool runs as a stand alone program It can be started from the software by choosing Tools gt Data Evaluation or can be chosen from the Agilent 2100 bioanalyzer program group by selecting Data Evaluation FS Agilent 2100 Bioanalyzer Contents A129 V Index Loading Electropherograms in the Data Evaluation Tool When you start the data evaluation tool from within the Agil
42. 7 V Index Principles of Nucleic Acid and Protein Sizing ona Chip Each chip contains an interconnected set of gel filled channels that allow for molecular sieving of nucleic acids or protein samples A series of electrodes control sample movement within the chip These make contact with the samples when the instrument lid is closed Each electrode is connected to an independent power supply providing maximum control and flexibility The electrode cartridge is also removable providing the flexibility to implement different configurations depending on the design of a chip A pressure cartridge is availble for cell based applications Microchannels are fabricated in glass to create interconnected networks of fluid reservoirs and pathways Contents A 228 V Index Glossary electrokinetic forces Electrokinetic forces are used to move switch and separate the samples Active control over voltage gradients directs the movement of materials using the phenomenon of electrophoretic flow electrophoretic flow A macroscopic phenomenon that results from an electrical double layer formed by ions in the fluid and surface electrical charges immobilized on the capillary walls When an electric field is applied the bulk solution moves towards one of the electrodes cathode Electrodes sit in the reservoirs that connect to the ends of the various channels Electrode potentials are applied to the various reservoirs in a time dependent fashion to move the f
43. DNA 1000_00000_2001 10 01_14 32 01 cld This dialog box appears when you choose Save As from the File menu It allows you to save a data file under a new or different filename You can also save as the same filename a dialog box will ask if you want to overwrite the old file The file can also be saved under the same or a different name to a different location on your computer Contents A219 V Index Save Selected Wells Dialog Box Save Selected Wells as x Demo DNA 500 14 PCR Mix 1 2iv PcRMx2 O O O OOOO O O ar a aaf PCR Mix 1 5 Iv JPCR Mix 2 6M fecavs SCS TL PcRMx3 tt s S sSCSY Bf PCR Mix 2 S IPCR Mix 1 WM PCRM WE Porm SCS Pr fram SSCS LW lade This dialog box appears when you choose Save Selected Wells from the File menu It allows you to save a data file of the selected wells under a new or different name When you click Save the Save As dialog box opens which lets you specify the filename and directory for data storage Contents A 220 V Index Tips and Shortcuts Keyboard Shortcuts Commands windows or dialog boxes can be accessed by selecting them from menus but the same items can be activated by keystroke combinations or by clicking buttons on a tool bar Description Menu Name Keyboard Shortcut ALT Key Shortcut File Menu ALT F Open CTRL 0 ALT F 0 Save CTRL S ALT F S Save As ALT F A Export ALT F E Upload to Data Organizer ALT F U Batch Up
44. Display n Results Table Single Well n Small Gel Display Activates pop up menu for sizing of window and closing the applicatio Activates and Grap Activates and Grap Activates and Grap Activates n pop up menu with combination of items h menus pop up menu with combination of items h menus pop up menu with combination of items menus pop up menu with combination of items and Grap N menus CTRL Left Click Left Mouse Button CTRL Key rom the Tools rom the Tools rom the Tools rom the Tools In a single well view in the large display overlays second and subsequent well data over original well data for each CTRL click on a lane in the small gel Each set of peaks is shown in a different color and line style Contents A 226 V Index Information about Your Computer A System Info feature has been shipped and installed with the Agilent 2100 Bioanalyzer software This feature can be used to examine your computer and show information about the operating system fonts printing screen display and more This information may be useful to the technical support representative if you call for assistance To view information using the System Info feature 1 On the Help menu click About Agilent 2100 Bioanalyzer 2 Click System Info 3 Click a category for the type of information you want Tip You can save or print information from the System Info dialog box Contents A 22
45. Electrode Cartridge Maintenance for more information regarding the use of the electrode cleaner and or the procedures for cleaning and or decontamination Decontamination 1 Slowly fill an electrode cleaner with 350 uL RNAseZAP in one well all wells are connected Label this electrode cleaner for RNAse ZAP 2 Open the lid place the electrode cleaner in the instrument and close the lid for approximately 1 minute 3 Open the lid remove the RNAse ZAP electrode cleaner and store it for future use You can reuse this electrode cleaner for all the chips in the kit Empty the electrode cleaner for overnight storage 4 Then follow the instructions below for cleaning the electrodes Cleaning after each run 1 Slowly fill another electrode cleaner with 350 uL RNAse free water in one well all wells are connected Label this electrode cleaner RNAse free water 2 Open the lid load this electrode cleaner into the instrument and close the lid immersing the electrodes in the water 3 After approximately 10 seconds remove the electrode cleaner Put this electrode cleaner aside for future use as well 4 Wait another 10 seconds for the water on the electrodes to evaporate Contents A29 V7 Index Preparing and Running an Assay DNA RNA and Protein Assays Check that you have everything listed in the appropriate Reagent Kit Guide Be aware that there can be small but important differences between the differen
46. Index
47. OTE The maximum number of samples the data evaluation tool allows you to display in the graph window is 12 No axis scales for example time in seconds for the x axis are shown in the display Contents A 132 V Index Alignment of Electropherograms in the Data Evaluation Tool Principles of Alignment Within the data evaluation tool the term alignment describes the process of making two or more electropherograms more comparable by stretching or compacting them in either or both the X and Y axis directions The data evaluation tool does this by using two reference points on each sample trace and then aligning these points on the traces When aligning along either the X or Y axis the first point defines where the alignment starts The distance between the first and the second reference point defines the scaling factor by which each curve will be stretched or compressed While it is possible to overlay and align electropherograms from any two or more runs the data evaluation tool works best with similar or identical samples It is useful for comparing RNA preps You may choose to use Automatic Alignment in which the data evaluation tool chooses the reference points and aligns both axes automatically Or if this is unsatisfactory you may use Manual Alignment instead which requires that you set the reference points for X and or Y axis alignment Automatic Alignment A sample can be aligned in three ways along the X axis along t
48. V Index The results are displayed in a tabular format d Re iew BEE Bio g_Total RNA_00000_2001 07 04_12 47 48 cld Assay Demo Eukaryote Total RNA Read 04 07 01 12 47 47 Data Path _C Programme Agilent 2100 Bioanalyzer Bio Modified 04 07 01 12 49 37 Ladder Total RNA Area 122 08 RNA Concentration 150 Peak Cor Area 1 27 80 18 23 2 31 00 19 45 3 34 95 7 38 4 39 45 14 61 5 45 30 24 04 6l 50 10 572 7 50 55 651 Wellt mouse liver RNA 20 no til Conected RNA Area 1564 RNA Concentration 1921 RNA Ratio 285 185 1 89 Fragment Name Start Time s End Time s Area Z of total Ares 1 185 38 20 39 05 3 60 23 02 2 285 4375 4560 679 4344 Agilent Technologies 2100 Bioanalyzer Bio Sizing Version A 01 01 N A Click Print to send the combined results to the selected printer Contents A95 V Index Data Analysis Protein The data analysis process for Protein assays consists of the following steps 1 Raw data is read and stored by the system for all of the individual wells 2 The data is filtered and the resulting electropherograms of all wells are plotted You can change the settings of the filtering algorithm after the run and reanalyze your data 3 Peaks are identified for all wells and are tabulated by migration time You can change the settings of the peak find algorithm and reanaly
49. Well Above Up Arrow Select Well Below Down Arrow Select First Well Ladder Home Select Last Well End After Zooming In on a Plot Scroll Horizontally Left Right Arrow Scroll Vertically Up Down Arrow Page Horizontal Scroll Right Shift Page Up Page Horizontal Scroll Left Shift Page Down Undo zoom and show regular Home plot Contents A 224 V Index Mouse Shortcuts Click and Drag Mouse In Single Well Large Display zooms in to selected rectangle Single Left Mouse Button Left Click In Small Gel Selects a well In Multiwell Large Display Selects a well In Single Well Large Display RNA assays If on a long dashed line move start end times for that well only move start end points for individual peaks DNA assays if on a peak selects the peak shows highlighted in the Results Table and pointer appears over the peak n Chip Selects a well n Tool Bar Activates function associated with button in tool bar Double Left Mouse Button Left Double Click n Small Gel Goes to Single Well View for lane double clicked n Multiwell Large Display Goes to Single Well View for well double clicked n Chip Goes to Single Well View for well double clicked n Single Well Large Display Undoes last step of zoom Single Right Mouse Button Right Click n Small Gel Selects a well n Multiwell Large Display Selects a well Contents A225 V Index n Title Bar n Large Gel Display n Single Well Large Display n Multiwell Large
50. a file with the name that is shown above the data display Data File BioSizing_00000_2000 08 26_10 27 52 Read 8 2600 10 27 52 AM Contents Al6V Index 9 When the assay is finished the General Chip Information dialog box with the Chip Run Summary tab in the front will appear and a sound will alert you you can stop the sound by clicking the Turn sound off check box Remove the chip from the bioanalyzer and dispose of it according to the guidelines established by your laboratory safety officer i General Chip Information Lx File Information Study Information Samples Information Chip Run Summary vA Status Assay completed Read 05 07 01 16 09 32 Sample Observation vi 3 sample peak s found in NEB 1 kb ladder 14 sample peak s found in Ad2 Dra 10 sample peak found in Ad2 Bgl l 7 sample peak s found in Ad2 Pvu l 2 3 4 5 6 sample peak s found in High Mass Ladder 6 12 sample peak s found in Synthetic DNA Ladder 7 3 sample peak s found in NEB 1kb ladder 8 13 sample peak s found in Ad2 Dra l 3 Possibly no restriction digest 10 7 sample peak s found in Ad2 Pvu 6 sample peak s found in High Mass Ladder 12 12 sample peak found in Synthetic DNA Ladder SSSE 88558855 Export Run Log oK Cancel The dialog box shows the number of peaks DNA and Protein fragment ratios total RNA or percent of rRNA contamination mRNA found in each sample and any assay specifi
51. aa 13972 ao B 158 rio m w 102 15 n ms zaa ast 129 15 esio set wo a2 061 Upper Marker Printed 4 01 33024 PM Aent Tactesogies 2100 Bicansyzer Bo Sizing Version A0101 NIA Contents A144 V Index Printing a Combined Results Table Ladder Peak Pesk Peak ir Peak F Wig Time seon wos a10 805 mas 7400 7525 7705 25 e510 Well 4 Sample Mig Time wees 3700 Weli 2 Sample 2 Mig Time exes ped 5510 Well 3 Sample 3 Mig Time aeea pe S700 S00 pry EEJ mas 6515 7395 7525 7770 m25 mas 10 Well 4 Sample 4 Mig Time ecni B515 i0 Contents 4000 1809 3600 4300 Soo Era 50 00 700 4700 4500 ees ors 3215 Mea na Sto sra 208 Roo 3240 1558 129 So Saor pr as ins Sia een ao ar 2r sio aP 2 10 Soo 700 1000 1500 2000 Soon 7000 10380 s iP 2 siw mP 10380 ss 708 4m 2000 Sona 005 Ha soseo P 705 103m0 Cone ot a3 io io to 40 ory io i ot 33 52 gi fy t i 50 38 at 3i 29 ae a7 35 22 25 a eat 37 2 Molariy rnai rst CHI 20 1212 aos eo to 303 202 aar oat Moterity onan rst 7987 Motarity st et Molariy erst Tase an iat 1297 as 740 3 60 oer os oat Molariy emneul GE ao oat Observations Lower marter Upper Maree Observations Lower Markee Upper Mare Observations
52. ained by the Agilent 2100 Bioanalyzer software 1 a System Log which maintains a running record of all events that occurred with the Agilent 2100 Bioanalyzer while it is online with the Agilent 2100 Bioanalyzer software including the dates and times that the system went on and offline the version history etc 2 a Run Log which includes information about the particular run including the date and time of the run any problems that occurred the assay that was used to generate the data and the name for the saved data file An example of information stored in a Run Log might look like this 04 30 1999 07 57 37 Start Data C PROGRAM FILES HP BIO SIZING Assays DNA DNA7500 asy C PROGRAM FILES HP BIO SIZING Data Bio Sizing_00329_1999 04 30_09 57 37 cld 04 30 1999 10 02 12 End Data 13 wells read 0 1234567891011 12 3 a Diagnose Log 4 an Installation Qualification Log Options Opens the Options dialog box which contains four tabs Data Files Reader Chip Alert and Advanced For more detail see Options Dialog Box on page 205 Contents A195 V Index Help Menu Help and Index About Bio Sizing Contents and Index Opens the Contents Index page for this Help function About 2100 Bio Sizing Opens the About box showing the Agilent 2100 Bioanalyzer software version number the Agilent 2100 Bioanalyzer software authors and so on Start Dialog Box File Prefix BioSizing Assay re DNA 7500 Smear
53. all data for the run has been collected To see a single well view either select a well and click the single well display button in the tool bar or double click the desired well in the multiwell view You can change the names of the sample wells to any names you wish To change a name highlight the existing name and type over it When the file is saved the new well names will be saved with the file Contents A 175 V Index Large Single Well Graph View PCR Mix 2 Fluorescence Sample Settings Resuits Enors Name 2 3 PRm O 3 Comment 5 50 158 200 210 300 315 400 420 450 6 3736p 7 73 j s 8 8785 7o 38 47 jena TV Rest Digest Exp Fragment BP 3 8365 aa 3o as 1390 an si7n amo oa ae tami zil Ease coca When the large display shows a single well you can zoom in to see the data in the graph more closely Drag the mouse in a rectangle that bounds the area you wish to view in more detail This area will enlarge to fill the large display area Unzoom by double clicking or use the unzoom tools The dividing line between the graph and the Results Table and the electropherogram display can be moved in the vertical direction giving more or less space to the Result Table This allows you to view all of the results at once for example in wells having several peaks Contents A176 V Index You can also overlay the graphs from more than one well in a single well d
54. ams Data from multiple wells can be overlaid within the single well large display view Hold down the CTRL key and then click the left mouse button on other lanes in the gel like image in the lower left corner of the screen Ad2 Bglll Ad2 Bgl Il Synthetic DNA Ladder Fluorescence es 0 U 56 5 Mm 6 m 5s w Time seconds Contents A52V Index Bounding boxes will appear around the gel lanes signifying which electropherograms are shown overlaid Li23456789 0N 12 Only ladder selected Ladder with sample 1 overlaid Each peak graph will be shown in a different color and line style with a legend at the top of the window You can remove wells from the overlay by CTRL clicking the corresponding lane in the small gel display the bounding box will disappear Contents A53V Index A stand alone program called Data Evaluation is also included with the bioanalyzer software and can be accessed by choosing Tools gt Compare Results This program allows you to compare the results from the same or different runs and even different assays within a single window and provides tools that allow you to manipulate the comparison of the data in different ways Documentation and help for the Data Evaluation program are available within the bioanalyzer software Contents AblV Index Changing to Gel View To see an overview of your data in a gel like image switch to the Gel view In the menu bar click
55. analyzer Software Screen Menu Bar File and Assay Information Area Tool se adea Bias Di He ain DuA 120m eT TERED Rae eee NED Tokia menei ranan Agilent 2100 3 Bioanalyzer omen i coll i Ma Chip Icon kPa Highs Leal Shete DNA Lae E d L o NEB kb ladder Ad2 Dial Ac2 Balll dA a BY atk AANA M Vee PEN Mi Li mn Error Information 2 Fea i Bie Peno Mode AUDENOT AUDFIN g Status Line Small Display Large Display Contents A 166 V Index The main screen of the Agilent 2100 Bioanalyzer software includes a title bar menu bar tool bar Agilent 2100 Bioanalyzer icon file information large display area small display area error area and status line These elements are described below When you first load the Agilent 2100 Bioanalyzer software the default view is in a window that fills the screen You can size this window smaller the Agilent 2100 Bioanalyzer software will remember the window size and position and will restore the settings the next time you start the software Title Bar Agilent Technologies 2100 Bioanalyzer Bio Sizing BioSizing_DNA 12000_00000_2001 07 05_16 09 3 The title bar extends across the top of the window inside the window borders It displays the name of the file and indicates whether it is the active window or not The title bar of an ac
56. and time of the run files would bear names such as Bio Sizing_2000 05 03_14 09 12 Contents A 205 V Index NOTE If you choose not to use the time of the run as part of the data filename the Agilent 2100 Bioanalyzer software will automatically append a 1 2 and so on for each subsequent run made that day This tab also allows you to choose the directory in which data files will be stored The default stores them in a directory with the software files but you can create and use a custom directory if desired You can also choose to have daily subdirectories created for file storage Saved files can be altered and resaved or saved to a different name if desired Contents A 206 V Index Reader Tab Data Files Chip Alert Advanced Serial Port None Z OK Cancel Serial Port This setting allows you to choose the serial Com port to which the Agilent 2100 Bioanalyzer is connected Com1 through Com16 A setting of None is also available if you are running the Agilent 2100 Bioanalyzer software alone without a connection to an Agilent 2100 Bioanalyzer Contents A207 V Index Chip Alert Tab i Options x Data Files Reader Alert sound C Off Default sound Custom sound C ProgrammeAgilent 2100 Bioanalyzer Bic El Alert interval seconds E E Cicecic cece 1 15 The settings under this tab have to do with the alert sound that is made when a chip needs to be removed from the Agilen
57. ange All Wells to combine the results of all measured wells Wells to combine the results of selected wells Options Exclude Markers to display the values without the markers Include Ladder to display the values of the ladder in a separate table Combined Result View x Well Range o C Wells Enter well number and or well ranges separated by commas Example 1 2 5 12 L for Ladder r Options I Exclude Markers I Include Ladder cores Contents A 126 V Index The results are displayed in a tabular format 200_00000_2001 07 04_12 54 41 cld Assay Demo Protein 200 Lysates Standards Read 04 07 01 12 54 41 Data Path _C Programme Agilent 2100 Bicanalyzer Bio Modified 04 07 01 12 57 24 Ladder Peak Mig Time secs Con Area Size KDa Rel Conc Observations 1 18 30 203 73 6 00 74 00 Lower Marker 2 19 65 120 66 9 00 74 00 3 21 00 112 50 14 30 74 00 4 22 05 137 32 18 40 74 00 5 23 55 11052 23 00 74 00 a 25 50 83 38 43 00 74 00 7 28 00 52 12 68 00 74 00 8 23 85 56 41 97 40 74 00 E 38 30 52 24 210 00 74 00 Upper Marker Welll Protein Standard 7 proteins Peak Mig Time secs Con Area Size KDa Rel Conc Total Observations 1 18 30 108 83 6 00 74 00 Lower Marker 2 19 45 4753 8 56 61 50 System Peak 3 20 95 28 48 14 10 36 80 11 7
58. arge display will cause numbers to appear next to a crosshair pointer What is displayed depends on the type of assay selected e With a DNA assay you will see the base pair measurements for the area of the lane beneath the crosshair of the pointer shown by a If the cursor is positioned over a recognized band the cursor will change to show a target and the concentration and molarity With RNA assays the size estimate in terms of of nucleotides nt and over recognized bands the area and percent of total area is shown e With a Protein assay positioning the cursor anywhere in the gel image will show the size of the protein in kDa for the area of the lane beneath the crosshair of the pointer shown by a If the cursor is positioned over a recognized band the cursor will change to show a target and the concentration is also shown NOTE The display of the gel can be changed to a number of different color combinations These are selected using the arrow to the right of the gel button in the tool bar or via the View menu For more information see View Menu on page 187 Contents A 1817 Index Small Display Small Gel View This area of the workspace shows either a gel default view of the data received or a single well display if the large display is showing a gel view Contents A 182 Y Index One lane of the small gel view will be bounded with a box This is the selected lane which corresponds to a sele
59. as Example 1 2 5 12 L for Ladder Options r T Include Ladder Cancel This dialog box appears when you choose View gt Combined Results It provides options to generate a table view of combined results of the current measurement You can choose between a view of all wells and a selection of wells that you can specify You can define if markers should be excluded and if the ladder values should be included Contents A216 V Index Combined Results Dialog Box BioSizing_Protein 200_00000_2001 07 04_12 54 41 cld Assay Demo Protein 200 Lysates Standards Read 04 07 01 12 54 41 Data Path C Programme Agilent 2100 Bioanalyzer Bio Modified 04 07 01 12 57 24 Ladder Peak Mig Time secs Cor Area Size KDa Rel Conc Observations iM 18 30 203 73 6 00 74 00 Lower Marker 2 19 65 120 66 3 00 74 00 3 21 00 112 50 14 30 74 00 4 22 05 137 32 18 40 74 00 5 23 55 110 52 23 00 74 00 6 25 50 83 38 43 00 74 00 7 28 00 52 12 68 00 74 00 8 29 85 56 41 97 40 74 00 Ej 38 30 52 24 210 00 74 00 Upper Marker Welll Protein Standard 7 proteins Peak Mig Time secs Cor Area Size KDa Rel Conc Total Observations 18 18 30 108 83 6 00 74 00 Lower Marker 2 1345 47 53 8 56 61 50 System Peak 3 20 95 28 48 14 10 36 80 11 74 4 22 45 1727 21 23 22 307 12 5 23 55 53 38 23 00 69 10 2
60. ateau This setting rejects brief low slope areas such as those found between non baseline resolved peaks Default enabled This setting causes the bioanalyzer software to use he values defined by the assay for ladder data instead of data obtained rom the ladder run with the assay Contents A4V Index You can change all peak find settings except the Baseline Plateau for individual wells In the lower right pane of the single well display to the right of the Results Table are four tabs The Settings tab shows the peak find settings that are currently in effect for that well Changing the settings shown on this tab will affect this well only to change the settings that affect all wells click the Global button to open the Assay Properties dialog box and then click the Global Peak Find tab Sample Settings Results Enors Min Peak Height 10 0 Global Min Peak Width 05 Reset Slope Threshold 0 8 Cancel Apply If you change the Global Peak Find settings after making individual well setting changes the following prompt will appear 7 Do you want to override custom well settings te ca Contents AMV Index Choosing Yes causes any changes made to the peak find settings for individual wells to be discarded and applies the global peak find settings to all wells Choosing No allows individual wells to retain changed peak find settings NOTE This prompt appears whenever at
61. ay allows you to move backward and forward through the wells Contents A170 V Index Agilent 2100 Bioanalyzer Chip Icon The Agilent 2100 Bioanalyzer or chip is represented on the left side of the screen What is shown in this display depends upon whether or not the lid to the Agilent 2100 Bioanalyzer is open or closed or a chip has been inserted When the lid of the Agilent 2100 Bioanalyzer is open the picture on the screen will show an open door letting you know that a chip has not been inserted and the lid has not been closed The picture will change to a closed door when you have closed the lid but no chip has been inserted The icon will appear dimmed if the instrument is switched off or is not connected to the PC Lid open Lid closed but no Dimmed icon chip inserted Instrument switched off or not connected to PC Contents AI1NV Index When a chip is inserted in the Agilent 2100 Bioanalyzer and you close the lid the icon will change to show the chip depending on the assay selected g Ei H 2 DNA LabChip Zs RNA Labthip Chip inserted and lid closed The chip icon is more than just a picture the currently selected well has a circle around it click a different well on the chip icon and the rest of the displays will update to reflect the new well choice During a run the white spot in the center of the well that is currently being assayed will blink File and Assay Information Area The file and assay
62. c messages such as results from a PCR fragment check Any errors associated with the run will also be shown You can view the Run Log by clicking the button at the bottom of the dialog box NOTE You can view the General Chip Information dialog box at any time by choosing Chip Run Summary from the View menu Contents A17V Index 10 Follow the cleaning protocol for the particular assay you were running as described in the appropriate Reagent Kit Guide 11 To view results for individual wells as data is acquired or after the run is finished click a well in he chip icon a single well on the large multiwell display or a lane in the gel image When you view the single well display specific data for that well appears in a Results Table at the bottom of the window 12 The bioanalyzer software can be set to print customized results automatically at the end of the run see Printing a Report on page 140 for more information You can also choose to print a report manually which can contain different information settings for the automatic and manual print functions are maintained separately 13 The bioanalyzer software can be set to export data automatically at the end of the run Settings for the automatic export function are customizable see Exporting Data on page 149 for more information You can also choose to export different information settings for the automatic and manual export functions are maintained separately and both are remembe
63. chip Each electrode in the cartridge is powered by its own power supply Different electrode cartridges can be used for different types of assays and correspond to different types of chips The chip receptacle is the recessed space that is designed to hold the chip in place The receptacle is keyed to the chip so that you cannot insert the chip improperly The chip selector ensures that only the right type of chip is used with the right type of cartridge position 1 applies to all kinds of molecular assays DNA RNA and protein After inserting the chip and closing the lid the Agilent 2100 Bioanalyzer icon changes to show a chip that looks like this oO 2 E Ei 3 3 2 HB DNA LabChip RNA Labthip NOTE If the icon did not change to show a chip then the chip is not detected see Maintenance and Troubleshooting Guide Contents Al4V Index 6 Click the Start button located above the chip icon The Start dialog box will open You can change the File Prefix used as the beginning of the saved filename and or enter notes about the run File Prefix BioSizing Assay p mRNA Nano Run sample 1 to BS I Edit samples after start caa 7 Click the Start button on the Start dialog box The assay begins it will take approximately five minutes before data appears on the screen The status indicator on the front of the Agilent 2100 Bioanalyzer flashes during the assay and the chip icon and other d
64. ck the Add button 2 Enter the From and To parameters for the region 3 Click Apply Contents A 82 V Index chicken liver mRNA 38 37 36 7 Region 35 End time 34 33 32 Fluorescence 31 30 29 28 Baseline 27 ji 4 4 4 4 4 4 7 22 27 32 37 42 47 52 57 62 67 72 Time seconds 7 The results related to the defined region are displayed in the Results table If you have defined more than one region you can select the desired region in the Results table From Lower limit of the region in b To Upper limit of the region in b Corr Area The area under the peak within the region of total Area Percent of the total area which is defined by the start and end time markers Observation Additional information about the peak Contents A83V Index Global regions can be defined by choosing Assay gt Assay Properties and selecting the Regions tab You can enter the lower and upper limit of the range and the range s color f Demo mRNA Smear Properties xi Summary Ladder Analysis Global Peak Find Regions From b To b g lor 000 1 foo 2 foo o TUUU LLLLLLLLEN Contents A84v Index Changing the View of the Results A number of different options are available for viewing the data after it has been acquired by the Agilent 2100 bioanalyzer None of these options change the raw data but rather pr
65. cseeseesesseessesssussuessseesnssneseesessessneeees 138 Printing a Report 0 Printing an Assay Summary Report 2 Printing a GeliR port sci cs cesta San a oes ced acon ceaneieece acaden use ctu aie 143 Printing an Electrophero grains sci cossscsey ccesectcnsedssctesss ccbuecseiceenicts E ERRE 144 Printing a Combined Results Table cccecccsscssessessessesseesessessesseeseesessesesstssessuestsseesneeseseesee 145 Printing 4 wells per page Electropherogram ccceccccccssessessessessessesseeeessessseestsseesteseeseesee 146 Setting Up Your Printer c ccccccccccessccsessessnesssseeseesesseeseesessecsesseesesseeseesesusesessesussnesseessessneseeses 147 Exporting Data 9 Uploading Chip Data to the Data Organizer Copying Information Usmo Helsen a EENAA Contents and Index o oo eeseecseecsessseesseeseesnecssesneessesseesuecsscesecssseueesnesueesneesneeneesesseesneeseesneeeeesneeeeee Context Sensitive Help ececccccccescsessessessesessssesseesessesssseeseesesssssecseeseesesseesssnesusesseessnseneseeeeeeee Primtinng Help sssicscsacesssdsasiccssaidstedes saniainen iarten asebczletseadsOhiabbatesssestantshtdeptatsetedesbianeadisien Types of Help Available cccccccccecsessscsessesssesesseesnesecssesesssscscsessesssesesuesusescsuseussnesteseesesases Mouse Notation Conventions in Help Keyboard Notation Conventions in Help Contents ASV Index Specifications iiisccicsesicsicsacciadc
66. cted well in the chip icon or in the multiwell display Clicking on a different lane will cause that lane to be selected and if a single well is shown in the large display the large display will update to show the currently selected well data When a new run is begun the display will be blank and the first lane ladder well will be selected As data is acquired in lanes wells the selection rectangle around the lane will increment to show the lane that is currently acquiring data If you select a lane well that is earlier in sequence than the current well however the display will no longer increment as new data is acquired but will remain on the selected lane well Small Graph View When a gel view is selected in the large display the small display will show a single well view of the data in the selected well Sample 2 Contents A 183 V Index Error Information Al Marker peaks in Sample C3 cannot be aligned to the ladder The area of the screen just above the status line is where most error messages will appear NOTE Click Al for context sensitive help Error messages can result from hardware or software problems Most are the result of peaks not being located by the analysis algorithms of the software This can be due to a sample peak or ladder peak not appearing as expected the settings in the software via the Peak Find Settings dialog box can also cause peaks to go undetected which can cause errors Additionally manua
67. d Cyd Labeled Nucleic Acid Nano Eukaryote Total RNA mRNA Prokaryote Total RNA Smear DNA 12000 Smear DNA 7500 Smear mRNA Smear Nano mRNA Smear Protein 200 Assay Properties Open Assay Contents These assay can be used to analyze Cy5 labeled cRNAs and cDNAs before hybridizing these samples to microarrays A rough size estimate can be obtained and failed cDNA labeling reactions can be identified These assays can be used to analyze specific smear DNA and RNA probes Using these assays makes additional evaluation methods such as regions available The Protein assay included with the bioanalyzer software is designed to analyze multiple types of proteins such as column ractions cell lysates and purified proteins This menu shows the name of the current assay Choosing this command opens the Assay Properties dialog box which displays he settings used to determine the ladder peaks and other settings required for analysis The settings on the first three tabs of this dialog box are not changeable by the user Settings on he last tab Peak Find are changeable see Peak Find Settings or more information Brings up the Open dialog box allowing you to open an assay hat is not currently shown in the Assays menu residing in another location A 192 V Index Save Assay Save Assay As Start Choosing this command will save an assay file with updated properties This command is dimmed if the current assay has not
68. d chip data to the central Data Organizer repository Batch Upload to Data Available only if the Data Organizer Client is installed Allows you to Organizer select a list of files and upload them to the central Data Organizer repository Print Opens the Print dialog box allowing you to choose the items that will be sent to the printer Page Setup Opens the Page Setup dialog box allowing you to change page orientation and margins lt File name gt Properties Opens the General Chip Information dialog where you can enter study information and sample settings for the currently loaded file 1 10 Recent Files The ten data files that were opened most recently the most recent file is labeled 1 Exit Allows you to close the software If you have unsaved data you will first be asked if you want to save it Edit Menu Edt Copy Gel Copy Graph Copy Results Table The copy functions in this menu allow you to copy a gel graph or a result table for use with another software Contents A 186 V Index View Menu View E Single Well F2 All Wells F3 E View Get F4 Combined Results 4 Previous Well F5 gt Next Well F r J E Black on White Standard Curve White on Black Calibration Curve i Green on Black Magenta on White Gl Red on Black E Pseudo on black fy Chip Run Summary Single Well Shows the single well view of the selected well in the large display All Wells Shows all of the wells in
69. dard curve of migration time versus size is plotted from the DNA RNA or Protein sizing ladder by linear interpolation The standard curve derived from the data of the ladder well should resemble the one shown above Contents A 203 V Index Calibration Curve Dialog Box sf Calibration Curve ol x Y O06 R 2 0 9547212 Re tte Corc gAn p Ha 8 8 0o m w mw a w a w m m m Serda Conc gmi If a standard protein was added on the chip and you have selected the option Use for Calibration on the Sample tab a calibration curve is calculated See the figure above for an example You can display and print the curve by clicking View gt Calibration Curve This dialog box is only available for protein assays Contents A204 V7 Options Dialog Box The Options dialog box contains four tabs Data Files Reader Chip Alert and Advanced Data Files Tab i si Reader Chip Alert Advanced Create data file names by combining I Prefix BioSzinn IV Assay Class I Serial Number MV Date M Time Example Bio_Sizing_DN amp 7500 SmearSRNUM_2001 07 05 Data file directory Default C Programme Data Data File settings allow you to determine the way in which data files are automatically named You can include a prefix of your choice the assay class the serial number of the Agilent 2100 Bioanalyzer the date and or the time of the run For example including a prefix such as Bio Sizing as well as the date
70. data first The items that can be changed for reanalysis are e Global Peak Find settings Individual sample peak find settings chosen in the sample information pane to the right of the Results Table in the single well view window see Settings Tab Gel color Sample names and comments e Exclude peaks from analysis e Reassign upper lower markers Alignment or no alignment with ladder peaks Contents A124 V7 Index Baseline Correction e Calibration Use of ladder run with samples or use of internal assay ladder Assay you can save the changed settings under a new assay name if desired NOTE If you save the data file after making changes it will keep a record of the assay if a new assay name has been saved it will use the settings from this assay the next time the file is opened gel color well names and peak find settings that were in effect at the time the file is resaved If you do not want to change the original file choose Save As and give the file a new name or save it to a different location Combining Results If you want to combine the results of different wells you can select the wells with the results to be combined You then can print a table view of the results To do this 1 Click View gt Combined Results 2 Select the wells to be combined 3 Click OK to display the combined results in a table view The items that can be changed for combining results Contents A125 V Index Well r
71. der peaks are analyzed to calculate a marker peak threshold which is used to locate the marker peaks in the sample wells If the marker peaks found using this method fail to align with those of a sample the bioanalyzer software will use the minimum peak height threshold setting instead if this value is lower than the value for the marker peak For example the calculated threshold might be too high to find the sample s markers if they happen to be very small Either no markers will be found or the wrong peaks will be assumed to be markers and these may not align with the ladder markers Consequently the software attempts to use the minimum peak height threshold which if it is set low enough will locate the real markers allowing the sample to align While the actual peak times are those shown in the unaligned data the bioanalyzer software cannot perform analysis without alignment so relative migration times are used aligning the markers to the ladder peaks causes a shift in the rest of the peak times Contents A41V Index If you are not sure that the software has selected the correct markers you can set the markers manually 1 Switch off the marker alignment using the Turn Off On Analysis icon in the toolbar 2 Click in the gel image on the ladder lane and then click the Scale To Selected Well icon Now you see all marker bands with approximately the same intensity in the gel like image You can recognize a ce
72. detection occurs within the window area plus 15 on either side 100 ng rat brain mRNA Fluorescence Time seconds After detection the ratio of the two rRNA bands is calculated and displayed 6 To calculate the concentration of the RNA the area under the entire RNA electropherogram is determined and compared with the ladder area Contents A 68 V Index Changing Your Data Analysis RNA and RNA Smear Assays Changing the Settings of the Data Evaluation Algorithm Different sets of parameters can be changed in the software in order to modify the data evaluation for sample analysis Filtering parameters Peak find parameters for all wells peak height for individual wells Time window for analysis Setting the baseline Aligning the RNA samples e Adding or deleting fragments e Adding regions in smear assays Changes can be made to reanalyze the data of a run Filtering Parameters The first step the software takes in analyzing the raw data is to apply data filtering Highlighted in the following figure are the two filtering parameters that can be changed Polynomial Order and Filter Width Contents A 69 V Index One way you can access the Global Peak Find tab of the Assay Properties dialog box is by choosing Assay gt Assay Name Properties gt Global Peak Find Eukaryote Total RNA Nano Properties Contents A10V Index The Polynomial Order setting is
73. e Data Files cld 7 Cancel T Open as read only Help When you choose Open from the File menu this dialog box appears Choose a data filename from the list in the box and click the Open button or double click a filename to open that data file This dialog box contains a check box which opens the file as read only and Open Read Only is displayed after the filename in the title bar A read only file may be edited but may not be saved under the same filename If you attempt to save an edited read only file an error message will be displayed explaining that the file is a read only file Clicking Okay in the error box will open the Save As dialog box Entering the same name as the read only file causes another error message to be displayed telling you to save the file with a different name or to a different location Contents A202 Y Index The benefit of opening a file as read only is to prohibit you or other users from making changes that would alter the file in any way Since the Agilent 2100 Bioanalyzer software allows you to open data files reanalyze them using different assays alter peak find parameters etc and saves these new parameters with the file when it is saved you may prefer to ensure that the original parameters which were used to create the file are not altered Standard Curve Dialog Box Standard Curve olx Point to Point Fit ra Cuve FII Lader Fragmente 8 g E3 A stan
74. e not changeable Demo DNA 7500 Smear P x Summary Ladder Analysis Regions Min Peak Height 8 Reset Min Peak Width 05 Secs Slope Threshold 08 Sec Start Time 25 Secs EndTime 95 Secs Filter Width 1 Secs Baseline Plateau 0 5 Secs Polynomial Order 6 I Exclude Ladder use assay defined default values The Global Peak find tab allows you to make changes to the peak find settings apply them and if you are satisfied with the result save the changes you have made to a new assay file This assay file can be used with subsequent readings to generate new data files and will be called up if any such file is reopened Contents A212 V Index The choices on the Global Peak Find tab determine how peaks are detected and shown in the display Settings on this tab are user changeable and have the following functions Minimum Peak Height Minimum Peak Width Slope Threshold Start Time End Time Filter Width Contents Determines the limit below which a peak will not be detected When the Settings tab is displayed in the Sample Information area the single well display depicts the minimum peak height setting by means of horizontal green lines on the peaks Determines the limit in seconds under which a peak will not be detected This setting represents the amount of change in absorbance units over time required to indicate that a peak has occurred Changing this setting may cause certa
75. e slider on the right hand side lets you adjust the brightness Contents A 119V Index Moving the mouse pointer over a gel in the large display will cause numbers to appear next to a crosshair pointer With a protein assay positioning the cursor anywhere in the gel image will show the size of the protein in kDa for the area of the lane beneath the crosshair of the pointer shown by a If the cursor is positioned over a recognized band the cursor will change to show a target and the concentration will also be shown Different gel display colors are available by choosing View gt Gel Color and then choosing one of the color schemes from the drop down menu The colors are designed to approximate actual gel staining and imaging techniques Blue on white for example simulates a Coomassie gel often used with proteins The Pseudo color choice provides more detail 1 280 colors since it maps the signal into a larger color space than is available with the other monochrome options 256 levels of brightness View E Single wel F2 E Alwels F3 E View Gel F4 22 Combined Results 4 Previous Well F5 gt Next Well Fe PI J E Black on White L Standard Curve White on Black L Calibration Curve i Green on Black Magenta on White E Red on Black E Pseudo on black By Chip Run Summary Contents A 120 V Index Force Baseline to Zero Since all electropherograms show some amount of background fluorescence
76. e well data in a linear stretch or compression using a point to point fit 210 p 210 eee 117 p 117 p 374 97 4 66 7 pam pae 66 7 po 53 p o a5 2 5 144 144 _ 6 ee ee ee 6 OC Data before alignment of the markers Sample markers aligned to markers in ladder If the sample marker peaks are either more than twice as far apart or less than half as far apart as the ladder markers they are assumed to be the wrong peaks and analysis of the well stops producing the error Marker peaks not detected Contents A 111V Index NOTE With Protein assays the height of marker peaks is assay dependent Sample peaks are analyzed to calculate a marker peak threshold which is used to locate the marker peaks in the sample wells If the marker peaks found using this calculated method fail to align with those of a sample the bioanalyzer software will use the minimum peak height threshold setting instead if this value is lower than the value for the marker peak For example the calculated threshold might be too high to find the sample s markers if they happen to be very small Either no markers will be found or the wrong peaks will be assumed to be markers and these may not align with the ladder markers Consequently the software attempts to use the minimum peak height threshold which if it is set low enough will locate the real markers allowing the sample to ali
77. eicoiciiecsaisiasasediatioinsionsceresicasuiceictastaesdestaiestadiciisiecasniaitadinteeddeie 165 Software Reference acascccccacssccssscessasessceesesispasncerespeccencessessseteetartgrasssneentnncertenaesiencteeatecrctan 166 The Agilent 2100 Bioanalyzer Software Screen sccccssessesssesesseeseseeseesessecsecsessneseeseeseenees 166 karge DISA nenna ies teateds teste ssice erates acatntesard a eines eet 174 Small Display Menu Items Tips and Shortcuts Information about Your COMPUtET ccccessessessessessessesseesesseeseeseesessesseesessssusenestssessneeeeseesee 227 Principles of Nucleic Acid and Protein Sizing on a Chip 228 GLOSS ary siseasssiaccsscissiecccscesisncdesd ccScaus iecaesachosiseaseuacveied cotuasevesbaibivenene asdsisensaniesiennesacatenionsersesa Parts and Accessories About This Guide Did YOu KNOW sissessesacsecseacasesdedcaccsnauccs 243 244 Contents A10V Index Quick Step Overview 1 Make sure the Agilent 2100 Bioanalyzer is connected to line power and connected to the computer NOTE If the bioanalyzer is not connected to the computer refer to the printed instructions accompanying the instrument regarding how to set it up 2 Turn on the line switch at the rear panel The status indicator at the front of the Agilent 2100 Bioanalyzer comes on and shows green 3 Start the Agilent 2100 Bioanalyzer software After startup the Agilent 2100 Bioanalyzer icon on the screen show
78. ent 2100 bioanalyzer software the data file present in the software will be automatically loaded in the data evaluation tool If you start the data evaluation tool from the program group no data will be preloaded NOTE You do not have to close the Agilent 2100 bioanalyzer software before opening the data evaluation tool The software and the data evaluation tool are co executable Contents A 130 V Index The figure below shows the data evaluation tool interface HB Agilent Technologies 2100 Bioanalyzer Data Evaluation Bie View Chp Wel Grech Hep p lt a SSS akeo coall o F O a A A Sope Theshod 002 rterwiah 7 z EW Bio Sizing 00000_1999 10 26_11 mm Sample 1 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Graph Sample 11 Sample 12 Window Ladder File List Fluorescence Well Preview IM ere ae 4 eae Migration Time Load all data files holding samples you want to compare to by going to File gt Open in the data evaluation tool interface Contents A 131 V7 Index NOTE No limit for the maximum number of data files you can load is given in the software The amount of computer RAM available to the data evaluation tool limits the number of data files the software can handle In order to compare samples load the samples in the graph window by double clicking on the individual sample names in the file list N
79. ent is turned on again A3V Index 7 The standard curve plotting migration time against DNA size in conjunction with the markers is then used to calculate DNA fragment sizes for each well from the migration times measured 8 To calculate the concentration of the individual DNA fragments of all sample wells the upper marker is used NOTE The software allows you to define upper and lower markers yourself However a change in the selection of the markers will lead to quantitative changes of the calibration procedure and will therefore alter the entire data evaluation Contents A35V Index Changing Your Data Analysis DNA and DNA Smear Assays Changing the Settings of the Data Evaluation Algorithm Different sets of parameters can be changed in the software in order to alter the data evaluation for sample analysis Filtering parameters lt Peak find parameters for all wells peak height for individual wells Time window for analysis Assigning upper and lower marker peaks lt Aligning or unaligning marker peaks e Defining regions of interest for smear assays Filtering Parameters The first step the software takes in analyzing the data is to apply data filtering Highlighted in the following figure are the two filtering parameters that can be changed Polynomial Order and Filter Width Contents A 36 V Index You can access the Global Peak Find settings in the software by going to Assay gt Assay Properties and ch
80. er well causes this pop up menu to appear X Exclude Peak From Ladder Copy Results Table Right clicking an entry of the result table of a samp e well causes this pop up menu to appear da Manually Set Lower Marker AP Manually Set Upper Marker X Exclude Peak Copy Results Table NOTE Excluding a peak or manually setting a peak to be an upper or lower marker may cause errors in analysis You can move the boundary between the Results Table and the well graph up or down to increase or reduce the amount of space allotted to the Results Table making it possible to see all of the results at once Contents A4 V Index Aligning or Unaligning the Marker Peaks The upper and lower markers are then aligned to the ladder markers by resampling the well data in a linear stretch or compression using a point to point fit 600 0 OO i i a 500 500 p a 400 H 400 p 300 lt 1 250 250 p 20 200 a 150 ed 100 100 50 50 5 rm ca fe Data before using alignment markers Markers aligned to ladder If the sample marker peaks are either more than twice as far apart or less than half as far apart as the ladder markers they are assumed to be the wrong peaks and analysis of the well stops producing the error Marker peaks not detected Contents A4V Index NOTE With DNA assays the height of marker peaks is assay dependent Lad
81. es 3 g 1 Ready fine Demo Modo Ane Erpat Ane PrE 77 Contents A76 Index Move any other start or end points the same way The boarders can not be moved across the boarders of a neighboring peak NOTE Changing the start or end points of the fragment will change the calculated rRNA ratio Time Window for Analysis The Start Time and End Time parameters on the Global Peak Find tab see figure below define the time window within which peaks will be found X Eukaryote Total RNA Nano Properties x Summary Ladder Analysis Min Peak Height 0 5 Min Peak Width Secs 0 5 Reset Slope Threshold Sec 0 8 Start Time Secs 20 End Time Secs 69 Filter Width Secs 1 Baseline Plateau Secs 0 5 Polynomial Order 6 M Lower Marker Present IV RNA Alignment Save As OK Cancel Contents A718 V Index Setting the Baseline for Calculation of RNA Concentration At low signal to noise ratios the baseline that defines the area used for calculating the concentration of RNA assays is highly dependent on the settings for the Start and End Time You can adjust the Start and End Times thereby adjusting the baseline manually to ensure a good result even at very low signal to noise ratios Choose a single well view Two vertical long dashed lines indicating the set points for the Start and End times with the baseline drawn between them are displayed in the window 1
82. es will appear around the gel lanes signifying which wells are shown overlaid Each peak graph will be shown in a different color and line style with a legend at the top of the window You can remove wells from the overlay by CTRL clicking the corresponding lane in the small gel display the bounding box will disappear One sample selected Sample 9 with sample 8 overlaid Contents A117 V7 Index A stand alone program called Data Evaluation is also included within the bioanalyzer software and can be accessed by choosing Tools gt Compare Results This program allows you to compare the results from the same or different runs and even different assays within a single window and provides tools that allow you to manipulate the comparison of the data in different ways Documentation and help for the Data Evaluation program are available within that program Contents A118 V Index Changing to Gel View To see an overview of your data in a gel like image switch to the Gel view In the menu bar click on View gt View Gel The main window will change and display the results in a format as would be generated by a slab gel device Ladder HSA150 HSA300 HSA500 HSA1000 HSA2000 PBS IgG not IgG reducec Sizing Sizing ol reduced Standard A Standard B __ 10 KDa 100 0 ug ml 117 H Oe n S 53 m 325 w 21 5 w 14 4 S M LIL Th
83. esented on the electropherogram by diamond shaped points on the peak baseline in the same color as that shown in the RNA tab Dragging a diamond will change the start or end time and alter the baseline drawn between the diamond markers Area The area of the individual fragment measured in base pairs of Total Area The percentage of the area of the individual fragment compared to the total area or RNA measured above the baseline Observations Additional information about the fragment Contents A91V Index Reanalyzing a Data File Occasionally you may wish to open and view or reanalyze a data file that was run and saved previously The raw data values are saved in the data file along with the analysis settings that were chosen for the run so that the data can be reanalyzed with different settings To do this 1 Click File gt Open 2 Choose the filename from the list of data files 3 Click OK If you have no unsaved data currently open the chosen file will open allowing you to view edit the results If you have unsaved data open a dialog box will ask if you want to save the current data first The items that can be changed for reanalysis are Global Peak Find settings Individual sample peak find settings chosen in the sample information pane to the right of the Results Table in the single well view window see Settings Tab on page 178 Gel color Sample names and comments e Fragment names and colors associated
84. gn Contents A112 V Index Using Calibration You can run standards for protein calibration with known concentrations in sample wells The software will calculate the calibrated concentration of the corresponding protein in all sample wells based on the calibration curve Select the Use for Calibration check box on the Sample tab or on the Samples Information tab General Chip Information dialog box and enter the concentration of the standard protein Sample Settings Results Errors Name lt _ Comment in PBS I Use for Calibration Cone ug mi 100 Edit Samples Cancel Epp The software will automatically use the peak with the largest area as calibration protein and it will be marked as such in the result table Observation column In the other sample wells the software will automatically calibrate the protein which corresponds in size with the calibration protein It will be marked as calibrated protein Additionally on the Sample Information tab of the General Chip Information dialog box you can define samples that you want to use as calibration standards and enter a concentration The calibration standard should be run in different concentrations to generate a calibration curve The Contents A 113V Index software will automatically produce this calibration curve based on these inputs to determine the actual concentration of samples within the same chip A remark is added to the observations c
85. h you wish to align To align manually along the X axis click the Align in X button on the Alignment tool bar En Or choose Graph gt Align in X To manually align the Y axis click the Align in Y button located on the Alignment tool bar EJ Or choose Graph gt Align in Y Contents A 135 V Index Peak Find Settings Two Peak Find settings are available in the data evaluation tool Slope Threshold 0 02 Slope Threshold Determines the sensitivity of the data evaluation tool to the start of a new peak Setting the Slope Threshold to a smaller number increases the sensitivity causing more peaks to be found Setting the Slope Threshold to a larger number reduces the sensitivity causing fewer peaks to be found The default setting for Slope Threshold is 0 02 Filter Width p b Filter Width Determines the number of data points used for calculating the peaks This value determines the width of a floating window that is aid over the data points Within this window the data evaluation tool calculates a derivation to determine if there is a peak Baseline Subtraction Can help when signal to noise ratios are very low and is enabled by default for all wells Baseline subtraction can be changed globally or for an individual well by clearing the check box on the Settings tab in the Sample Information area Contents A 136 V Index Data Handling and Printing Organizing Retrieving and Back
86. he Agilent 2100 Bioanalyzer software It includes the filename directory software and hardware version and the file format Contents A197 V Index Study Information Tab The Study Information tab is part of the General Chip Information dialog box General Chip Information LJ File Information Study Information Samples Infomation Chip Run Summary Study Name Study Comments Experimenter Laboratory Company Department Import Export Run Log Ok Cancel Use the text fields to enter significant information on the current study This will help you to identify the study Contents A 198 V Index Sample Information Tab When you enable the Edit samples after start check box in the Start dialog box another window will open when you click Start af General Chip Information Lx File Information Study Information Samples Information Chip Run Summa Sample Names ad2 Drat jAd2 Bgl IL Ad2 Pvu 119 149 641 815 NEB 1kb ladder JAd2 Dral JAd2 Bql Ih Ad2 Pvul 119 149 641 275 351 1 NEB 1 kb ladder 500 1000 1500 2000 3000 4000 275 351 1270 1547 1549 1757 1669 2065 2179 2828 3945 4555 High Mass Ladder 1000 2000 3000 4000 6000 10000 Synthetic DNA Ladde 175 252 395 523 500 1000 1500 20 1547 1549 1757 1669 2065 2179 2828 3945 4555 High Mass Ladder 1000 2000 3000 4000 6000 10
87. he Y axis or along both axes simultaneously The X axis uses reference points defined at the center points of the first and last peak on an electropherogram With most DNA and Protein samples these are usually the marker peaks with total RNA samples these correspond to the 18S and 28S ribosomal peaks for Contents A 133 V Index eukaryotic samples and the 16S and 23S ribosomal peaks for prokaryotic samples The Y axis uses reference points defined by the baseline and the apex of the first peak By modifying the Filter Width and Slope Threshold settings you can alter the results shown Manual Alignment In some cases the reference points that are defined automatically may not be adequate In this case you may define your own points using Manual Alignment Manual Alignment consists of first placing new reference points on a graph which can be done by clicking on any data point and then choosing alignment in the X axis or the Y axis You may define only two new reference points on each electropherogram you wish to align a starting point and an ending point If you place only one point another reference point will be defined automatically by the data evaluation tool Where the software places the second depends on whether you are aligning the X or Y axis and where you define the first point e If you are aligning in X and you place a single marker in the first half of the electropherogram measured from the start of the first peak to the end
88. he original parameters that were used to create the file are not altered Contents A 138 V Index Changes made to an assay file altered peak find settings for example can be saved to the same assay file or to a new one if desired Clicking the Save as button on the Assay Properties dialog box opens the Save As dialog box allowing you to save the assay Saving Selected Wells In addition to the regular file saving you can save a selection of wells Sometimes you may get wells with particular interest for yourself or you may want to exclude wells with poor quality For this purpose you can select specific wells and save them in a separate file 1 Choose File gt Save Selected Wells 2 Select the wells you want to be part of the file 3 Click Save and set the filename and directory in the dialog box Contents A 139 V Index Printing a Report Choosing File gt Print opens a dialog box which allows you to print up to four different types of information in a report from a data file r Print IV Assay Summary T GelLike I Electropherogram I Combined Result Tables r Well Range r Options E 4 per age Ge We fo Brelude Maken C wasl Be Inelude Ladder Enter well number and or well ranges separated by commas Example 1 2 5 12 L for Ladder You may choose one or more of the items shown by clicking in the check box next to the desired item Assay Summary and Gel Like always produce a
89. his Do this Click File gt Open Click the File menu with the mouse and click the Open item in the menu Click Assay gt DNA gt DNA7500 Click the Assay menu click DNA and click DNA7500 from the sub menu that appears Enable a check box Click the check box to place a check mark inside the box Disable a check box Click the check box to remove the check mark Select Click to highlight bound a well peak lane Choose Print from the File menu Click the File menu and click the mouse button on the Print command Right click and click Copy Click the right mouse button and click the Copy command from the submenu that appears Keyboard Notation Conventions in Help When you see this Do this Press Enter Press the ENTER key on your keyboard Ctrl Shift Press the Ctrl key and Shift key at the same time Ctrl A Press the Ctrl key and the A key at the same time Contents A 164V Index Specifications For physical specifications of the Agilent 2100 Bioanalyzer for example power consumption accepted humidity range and so on see the Site Preparation and Safety Manual that comes with the Agilent 2100 Bioanalyzer NOTE The specifications are subject to change without notice For the most recent specifications see the appropriate Reagent Kit Guide or visit the Lab on a Chip web site at http www agilent com chem labonachip Contents A 165 V Index Software Reference The Agilent 2100 Bio
90. ide Syringe Box 000 Kit hips reagents Kit Guide Syringe Box 500 Kit Chips reagents Kit Guide Syringe Box 2000 Kit Chips reagents Kit Guide Syringe Box Protein 200 Plus Kit Chips reagents Kit Guide Syringe Box RNA 6000 Nano Reagents Reagents 00 Reagents Reagents 200 Reagents Reagents 500 Reagents Reagents Protein 200 Plus Reagents Cooled Reagents Contents A 232 V 5065 4476 5064 8284 5065 4449 5064 8230 5064 8231 5065 4480 5065 4475 5065 4440 5065 4438 5065 4437 5065 4482 Index Accessories 1 Vortex Mixer Adapter for IKA vortexer 1 16 pin cartridge bayonet no extra electrode pin set pin set not re orderable 1 Chip Priming Station comprises 1 gasket kit 1 adjustable clip 1 TestChip Kit comprises 1 autofocus 1 Electrode Diode 5 Leak Current Clips Spare Parts 1 RS 232 Cable communication cable PC instrument 1 Fuse two fuses needed for G2938A 4 Gasket Kit comprises 1 plastic adapter 10 gaskets 1 Adjustable Clip for use with luer lock syringe d Multiport Cable for rocketport card Contents A 233 V 5022 2190 5065 4413 5065 4401 G2938 68100 62938 81605 2110 0007 G2938 68716 5042 1398 62938 81610 Index Index Symbols csv 150 tif 150 txt 150 xml 150 Numerics 12 well view icon 169 12 well view multiwell view 175 A accessories 233 Agilent 2100 bioanalyzer hardware
91. ighlighted in the following figure are the two filtering parameters that can be changed Polynomial Order and Filter Width Contents A101 V7 Index You can access the Global Peak Find settings in the software by going to Assay gt Assay Properties and choosing the Global Peak Find tab Demo Protein 200 Plus Properties x Summary Ladder Analysi Min Peak Height 2 Min Peak Width Secs 0 2 Reset Slope Threshold Sec 4 Start Time Secs 15 End Time Secs 47 Filter Width Secs 0 5 Baseline Plateau Secs 0 3 Polynomial Order 6 I Calibrate all Proteins M Baseline Correction IV Exclude Ladder keep current values Save As OK Cancel The Polynomial Order setting is used to define the power series applied to fit the raw data The higher the number you set the more the fit function will follow the noisy raw data curve As a result the noise level of the filtered curve will increase Contents A 102 V Index Filter Width defines the data window given in seconds used for averaging The broader the filter width the more raw data points are used for averaging As a result the noise level will decrease but peaks will become lower and broader Overall changing the Filter Width has more effect on the result of the filtering procedure that is applied than does changing the Polynomial Order Peak Find Parameters After data filtering the Peak Find algori
92. igration time is the amount of time from sample injection to the detection of a particular protein The area under the peak is corrected as a result of migration and baseline correction The peak size measured in kiloDaltons Relative protein concentration measured in micrograms per milliliter derived from the area conc relationship with the upper marker The concentration can only be given as relative concentration when comparing different proteins because dye binding differs from protein to protein Calibrated concentration of the calabration protein in the standard or of the calibrated protein in the sample Total The percentage of the area of the individual peak compared to the summed total area of all peaks in the sample not including markers and system peak Observations Additional information about the peak Contents A 123 V Index Reanalyzing a Data File Occasionally you may wish to open and view or reanalyze a data file that was run and saved previously The raw data values are saved in the data file along with the analysis settings that were chosen for the run so that the data can be reanalyzed with different settings To do this 1 Click File gt Open 2 Choose the filename from the list of data files 3 Click OK If you have no unsaved data currently open the chosen file will open allowing you to view edit the results If you have unsaved data open a dialog box will ask if you want to save the current
93. in peaks that were previously detected to be ignored or to interpret noise as peaks Shown on the single well display as a vertical green line this setting determines the time after the start of a run when the first peak can appear any peaks appearing before this time are ignored The vertical green line is shown as a solid line when markers are not aligned analysis off it is shown as a dotted line when markers are aligned This setting determines the time after which peak detection stops It is shown in the single well display as the end of the graph window scale This setting determines the width in seconds of the polynomial to be convolved with the data If you change the setting ensure that the value is less than twice the width of the peaks of interest or the peaks will be distorted A213 V Index Baseline Plateau Polynomial Order RNA Alignment Lower Marker Present Exclude Ladder Baseline Correction Calibrate all Proteins This is a baseline parameter for peak finding The signal is it baseline whenever the slope of the data is less than the slope threshold setting positive or negative for longer than the time specified for Baseline Plateau This setting is used to reject brief low slope areas such as at peaks in between non baseline resolved peaks This setting is used to define the power series applied to fit the raw data The higher the number you set the more the fit function will follow he noisy raw da
94. ing Up Data Files As you begin to work with the Agilent 2100 Bioanalyzer software it is good practice to organize your files If you are not the only user of the Agilent 2100 Bioanalyzer creating a directory within which to save your files is recommended having all users save their files to their own directories will speed the process of finding a particular file at some point in the future Even if only one person uses the Agilent 2100 Bioanalyzer software it is still wise to review your files periodically archive files you are no longer using but wish to save and discard unneeded files Each user in your laboratory may want to specify a particular file prefix which will easily differentiate their data files from any others Additionally you may specify that a new directory be created each day for storage of that day s runs It is a good idea to archive files to a backup disk for safekeeping and or to remove files from your hard disk periodically Depending on the amount of hard disk space available to the Agilent 2100 Bioanalyzer software you may need to clear space on your hard drive to ensure that you have enough room to save upcoming assay data Contents A 137V Index Saving Data and Assay Files File data is saved automatically at the end of a run Files are given a name that corresponds to the choices you have made regarding the prefix the assay name the serial number the date and time of the run see Data Files Tab on page 205 fo
95. ion 150 file information 173 file menu 185 filename 173 filter width 38 136 213 filtered curve 37 214 filtering algorithm 31 filtering parameters 36 101 RNA 69 filtering procedure 38 fit function 37 214 focusing lens cleaning 28 G gel brightness 56 copy 186 copying 159 gel color 188 gel display 181 gel electrophoresis 230 gel image exporting 149 gel mobility correction 210 gel report 143 Gel View 88 119 gel view 55 169 large 180 slider 56 Contents small 182 gel dye mixture 26 general chip information 197 global peak find 173 peak descriptors 213 settings 212 glossary 229 graph copy 186 copying 159 graph menu 189 graph view large multiwell 174 H handling tools chips and reagents 25 hardware 231 hardware checking 194 help contents 161 contents and index 196 mouse notations 164 printing 162 types of 163 help menu 196 help using 161 hotspots 163 A 231 V Index l Icon lid closed 11 icon chip 14 dimmed 11 lid open 11 K keyboard shortcuts 221 keyboard notation conventions 164 L Lab on a Chip 229 Lab on a Chip web site 25 laboratories on a microchip 230 ladder exclude 214 RNA 67 ladder well in gel display 181 ladder DNA 32 large display 166 174 single well graph 176 large display single well 175 large gel view 180 launcher 21 lens 13 lid 13 links 163 Contents liquid chromatography 230 local peak baselines 38 log file types 195 view 195 lower and uppe
96. ion algorithm sets the baseline to zero fluorescence units To disable the baseline correction deselect the Baseline Correction check box and click Apply Sample Settings Results Enrors MinPeakHeight 40 Global Min Peak Width 05 Reset Slope Threshold 05 Cancel Contents A59 V Index 110 Baseline correction disabled 105 100 D 9 o a A t 0 itis f o eousoses0n 4 Time seconds Baseline correction enabled busts t fi T o a fi T 0 fi T o e oueosei0n 4 fi T rr o Time seconds Index A60V Contents The Results Table The Results Table appears below the single well view in the large display area This table provides the following information Peak Number Mig Time seconds Area Size bp Conc ng uL Molarity nM Observations The order in which the peaks were detected The migration time is the amount of time from sample injection to the etection of a particular nucleic acid fragment a The area under the peak If the sample has been aligned the area of the igned peaks is reported o The size is the number of calculated DNA base pairs The concentration in nanograms per microliter for each fragment derived from the area conc relationship with the upper marker the same for all ladder peaks Concentration x 10 660 x Size
97. isplay To learn more about this feature see Graph Menu on page 189 Peak Number is the default for the single well display but other options are available Peak Area No Peak Info Peak Number Peak Height Peak Area Peak Size Peak Concentration Peak Molarity Sample Tab Sample Settings Results Enors Name PCR Mix 3 Comment 500 550 600 650 700 750 800 900 1000 bp I Restr Digest Exp Fragment BP Edit Samples Cancel oe Ease cores The Sample tab lets you view and or edit the name and comments of the sample viewed To edit names and comments of all samples or to add notes about the run click the Edit Samples button To learn more about it see page 199 Contents A177 V Index Settings Tab Sample Settings Results Errors Min Peak Height 8 0 Global Min Peak Width O05 M Rest 08 Slope Threshold Canes Boat Change the Peak Find Settings in the Se Sample Settings RNA Results Erors Min Peak Height 05 Global Min Peak Width 05 Reset Slope Threshold 08 Baseline Start Time 22 00 Secs EndTime 69 00 Secs ment Cancel Apply tings tab It lists the settings used to determine whether or not a peak will be kept for analysis Changing the settings will influence the results of this well only For RNA assays additionally you can se RNA Tab Results Enrors Fragment 1 Add Name 165 z Delete Sta
98. isplays on the screen are updated to show which well is being read Contents A15V Index Selecting Edit samples after start on the Start dialog box causes the General Chip Information dialog box with the Samples Information tab in the front to appear You can enter information in this dialog box during the chip run File Infomation Study Information Samples Information Chip Run Summary Sample Names p ion e 500 1000 1500 2000 3000 4000 m 0 5 Ad2 Dral 119 149 641 815 1195 2058 300 5 Ad2 Ba I 275 351 1270 1547 1549 1757 4 o 5 Well4 Ad2 Pvul 1669 2065 2179 2828 3945 4555 x a 5 Well5 High Mass Ladder 1000 2000 3000 4000 6000 10000 a 5 Well Synthetic DNA Ladde 175 252 395 523 651 818 1039 T o 5 Well7 NEB 1kb ladder 500 1000 1500 2000 3000 4000 E a 5 Well8 Ad2 Dra 119 149 641 815 1195 2058 x o 5 Welg Ad2 Bgi 1 275 351 1270 1547 1549 1757 4 o 5 Wel 10 Ad2 Pvul 1669 2065 2179 2828 3945 4555 A o 5 Wal 11 High Mass Ladder 1000 2000 3000 4000 6000 10000 o 5 Well 12 Synthetic DNA Ladde 175 252 395 523 651 818 1039 o 5 Reset Epa C __ Export Run Log ok Cancel You can change the names of the samples add comments expected basepair values define standards for protein calibration enter standard concentrations and or notes about the chip or run if desired 8 Data is saved to
99. ld ike to print Single Well View Switches to a view of the selected well in the large display Multiwell View Switches to a view of all of the wells in the large display Gel View Switches to a view of the gel in the large display Clicking the small arrow to the right of this button opens a drop down menu allowing you to choose the coloring of the gel image Analysis On Off Turns analysis alignment of markers to the ladder on or off toggles ote that the state of this setting is saved even when the Agilent 2100 Bioanalyzer is powered down until you change it again Scale to Selected Well Scales all peaks to the scale with all peaks visible of the selected well Scale All Wells Causes all of the data to be visible within the individual well windows by scaling each well to itself If you hold down the Shift key while clicking this button or choosing this command each well is scaled to the well data having the highest peak Unzoom Tool If you have zoomed in for a closer view in the Single Well view by clicking and dragging the mouse use this tool to zoom back step by step A 169 V Index a blue Peak Number gt Undo Zoom Completely Tool This tool will reset the standard view Rh Drop down menu showing labeling options for the single well view graph Show Hide Data Points Turns the display of data points on and off for he single well view Backward Forward On the single well displ
100. least one of the samples has different local settings Contents Ay Index Time Window for Analysis The Start Time and End Time parameters on the Global Peak Find tab see figure below define the time window within which peaks will be found DNA 7500 Properties xi Summary Ladder Analysis Min Peak Height 8 Reset Min Peak Width 0 5 Secs Slope Threshold 08 Sec Start Time 25 Secs End Time 95 Secs Filter Width 1 Secs Baseline Plateau 0 5 Secs Polynomial Order 6 I Exclude Ladder use assay defined default values Contents A437 Index Assigning Upper and Lower Marker Peaks For each sample the upper and lower marker peaks are assigned first and then the data is aligned so that the well markers match the ladder markers in time This allows the size and concentration of the sample peaks to be determined The first peak is assigned to be the lower marker and is then offset to match the lower marker in the ladder The upper marker is then assigned either to the last peak in the sample well or to the peak nearest to the ladder s upper marker See Aligning or Unaligning the Marker Peaks 46 for an example of assigned marker peaks If you see unexpected peaks in the ladder analysis or the markers are set incorrectly you can exclude peaks manually from the ladder or choose a peak to be used as a marker Contents AMV Index Right clicking an entry of the result table of a add
101. line Plateau Secs 05 Polynomial Order 6 M Lower Marker Present IV RNA Alignment Save As OK Cancel The Reset button sets the Global Peak Find values back to the factory settings Contents A712 V Index Min Peak Height Slope Threshold Min Peak Width Baseline Plateau RNA Alignment Lower Marker Present Determines the threshold for the peak find algorithm For each peak the difference between the start point value and the center point value local baseline must be greater than the Minimum Peak Height value Determines the difference in the slope that must occur in order for a peak to begin The inverse of this value is used to determine the peak end Determines the minimum amount of time that must have elapsed after he threshold was exceeded A parameter that assists in finding peaks The signal is recognized to be at baseline whenever the slope of the data is less than the Slope Threshold setting either positive or negative for longer than the time set for the Baseline Plateau This setting rejects brief low slope areas such as those found between non baseline resolved peaks Enabled in all nano assays disabled in all other RNA assays This setting causes the bioanalyzer software to align the signals of RNA samples to the lower or upper marker You can set the markers by using he context menu in the result table if the Settings tab is displayed Peak display Ribosomal bands can be
102. lly excluding a peak from analysis done in the result table can cause errors with the Peak Find algorithm Status Line Reading Ladder 15 of 130 seconds TTT The status line is found at the bottom of the Agilent 2100 Bioanalyzer software screen and displays information relevant to whatever is currently taking place in the Agilent 2100 Bioanalyzer When the software is ready to run an assay the Status Line will display Ready When you begin an assay it will show each step as it starts including the total amount of time that step will require and how much time remains for that step to complete A progress bar on the right side of the Status Line provides a graphical representation of the same information Additionally it displays whether or not Auto Export and Auto Print are activated Double clicking the different cells of the status line allows you to open the Options dialog box see Options Dialog Box on page 205 Contents A 184 7 Index Menu Items File Menu Open Save Save As Save Selected Wells Export Opens a previously saved data file Saves an unsaved or changed data file Saves a data file under a new name Opens the Save Selected Wells dialog box to select the wells to be saved Causes a dialog box to appear allowing you to choose the type of data that will be exported Upload to Data Organizer Available only if the Data Organizer Client is installed Uploads the currently loade
103. load to Data Organizer Print CTRL P ALT F P Page setup ALT F U File Properties ALT F T Exit ALT F X Edit Menu ALT E Copy Gel ALT E E Copy Graph ALT E G Contents A221 V Index Description Menu Name Keyboard Shortcut ALT Key Shortcut Copy Result Table ALT E R View Menu ALT V Single Well F2 or CTRL W ALT V S All Wells F1 ALT V A View Gel F4 ALT V G Previous Well F5 or Left Arrow ALT V P Next Well F6 or Right Arrow ALT V N Standard Curve ALT V C Calibration Curve ALT V C Chip Run Summary ALT V U Graph Menu ALT G Scale to Selected ALT G S Scale All ALT G A Undo Zoom CTRL Z ALT G Z Undo Zoom Completely ALT G U Show Data Points CTRL D ALT G D Copy Graph CTRL C ALT G C Contents A222 Y Index Description Menu Name Keyboard Shortcut ALT Key Shortcut Assay Menu ALT A dsDNA Menu ALT A D RNA ALT A R Protein ALT A P Other ALT A H Demos ALT A E Assay Properties ALT A T Open Assay ALT A 0 Save Assay ALT A V Save Assay As ALT A A Start ALT A S Tools Menu ALT T Turn On Off Analysis CTRL A ALT T A Diagnose Instrument ALT T D Compare Results ALT T C Temperature Monitor ALT T T View Log File ALT T V Run Log ALT T V R System Log ALT T V S Contents A 223V Index Description Menu Name Keyboard Shortcut ALT Key Shortcut Options ALT T 0 Help Menu ALT H Contents and Index CTRL H ALT H C About Bio Sizing ALT H A Selecting Wells Select
104. luid in the direction you desire it to go The gel filled channels of the LabChip devices do not exhibit a measurable flow because of dynamic channel coating and viscosity of the polymer matrix electrophoresis A standard technique of separating molecules on the basis of their charge to mass ratios An electric potential is applied across a capillary containing a sample in a fluid medium Positive molecules migrate to the cathode and negative molecules migrate to the anode at different speeds depending on the charge to mass ratios lab on a chip The downsizing of analytical techniques from lab scale to chip scale using techniques like electrophoresis chromatography sieving with fluorescence absorbance MS detection Contents A229 V Index e with a higher degree of automation integrating multiple steps of a complex protocol into a miniaturized system Virtually everything that is done in a laboratory can theoretically be done on a chip microfluidics The movement of liquids through micro fabricated structures by means of electrical fields or pressure vacuum holding the promise of greater functionality with significantly improved reliability e small glass or plastic devices with micro channels as experimental platform active control of fluids without moving parts on chip through miniature electrodes or pumps controlled by software scripts emulation of conventional liquid pumps valves dispensers reactors separatio
105. n systems etc capability of liquid transfer separation dilution reactions and more molecular separation techniques Processes such as gel electrophoresis liquid chromatography and capillary electrophoresis which can separate bimolecular organic substances from other compounds miniaturized laboratories on a microchip Expression used to describe lab on a chip technology Contents A 230 V Index Parts and Accessories The following parts are available for the Agilent 2100 Bioanalyzer Bundles For up to date details refer to http wadnts02 wad hp com off sc pages unsec bundlist htm 4 Agilent 2100 Bioanalyzer Single Instrument System G2940AA VL400 PC Instrument accessories printer vortexer Agilent 2100 Bioanalyzer Multi Instrument System G2942AA VL800 PC instrument accessories printer vortexer 4 Agilent 2100 Bioanalyzer Laptop System G2943AA Omnibook 6000 instrument accessories printer vortexer Hardware Software 4 Agilent 2100 Bioanalyzer G2938A comprises 1 chip priming station 1 testchip kit serial cable site amp safety manual setup poster I Agilent 2100 Bioanalyzer System Software G2941AA Contents A231 V7 Index Reagent Kits and Reagents m t oO oO C QA Q 0 O E C DNA5 c DNA 1 C DNA7 DNA 1 Cooled DNA 5 Cooled DNA 1 Cooled DNA 7 Cooled RNA 6000 Nano Kit hips reagents Kit Guide Syringe Box 00 Kit hips reagents Kit Gu
106. ng to Gel View To see an overview of your data in a gel like image switch to the Gel view In the menu bar click on View gt View Gel The main window will change and display the results in a format as it would be generated by a slab gel device Ladder 100ngrat SOng 200ng 100ngrat SOng 200ng 100ngrat SOng 200ng 100ngrat SOng 0ng bian bovine mouse bain bovine mouse bian bovine mouse bian bovine mouse RNA frea 6 71 of tota 10 62 The slider on the right hand side lets you adjust the brightness Contents A83 V Index Moving the mouse pointer over a gel in the large display will cause numbers to appear next to a crosshair pointer With RNA assays nothing is shown except over recognized bands where the area and percent of total area will be shown Different gel display colors are available by choosing View gt Gel Color and then choosing one of the color schemes from the drop down menu The colors are designed to approximate actual gel staining and imaging techniques Blue on white for example simulates a Coomassie gel often used with proteins The Pseudo color choice provides more detail 1 280 colors since it maps the signal into a larger color space than is available with the other monochrome options 256 levels of brightness View E Single Well F2 All Wells F3 E View Gel F4 Combined Results 4 Previous Well gt Next Well Yl Black on White Standard Curve White on Black Calibration
107. o Sizing Data File E BioSizing ONA 1000_00000_20 4 Click on Start Batch Upload The Update General Chip Comments dialog box appears If you want to change general chip information data such as Study Comments or Experimenter name Modify the respective field contents A 156 V Contents Index Select the Overwrite existing Entry check box for those fields you want to be updated during upload These fields will be updated for all files that are uploaded by the batch upload process To clear the list click Remove all Files 6 Click OK The following figure shows the start of the upload process Upload Progress Loading File You can stop the upload process by clicking Cancel 7 If the upload was successfully a message appears for example 2 Files successfully uploaded to Data Organizer If an error occurred a dialog box appears with an error message like the following Bio Sizing 1 Error s occured during batch processing of 3 files Errors have been written to the Data Organizer Log File Do you want to view the Log File now teal Click YES to view the log file or NO to return to bio sizing NOTE You can also view the log file by clicking Tools gt View Log File gt Data Organizer Contents A 158 V Index Copying Information The Edit menu offers three choices for copying information from the Agilent 2100 Bioanalyzer software for use with other applications
108. of the last peak the data evaluation tool assumes the point to be the starting reference point and automatically assigns the ending reference point If you are aligning in X and you place the marker in the second half of the electropherogram the data evaluation tool assumes this point to be the ending point and automatically assigns the starting reference point If you are aligning in Y and you place a single marker less than halfway up the first found peak the data evaluation tool assumes you are choosing a point for the baseline and automatically assigns the second point at the apex of the first peak If you are aligning in Y and you place the marker more than halfway up the first found peak the data evaluation tool assumes the marker to be the apex of the peak and assigns the baseline automatically Contents A 1347 Index After defining all reference points you may start the alignment process in either direction After alignment all manually defined markers are automatically deleted Placing markers for manual alignment often is easier if you can see the data points along the electropherogram Click the Show Data Points button located on the Options tool bar A Or enable Show Data Points on the View gt Options dialog box Any of the data points now showing may be selected as a new reference point The mouse cursor will change to a pointing hand when a data point may be selected Choose either one or two points on each grap
109. olumn to identify the calibration protein and the calibrated proteins General Chip Information xi File Information Study Information Custom DO Information Chip Run Summary Sample Name Sample Comment Use for Calibration Cone pg ml 4 2000 00 1000 K 1000 00 A 500 00 w 250 00 A 100 00 wv 25 00 BSA 1250 H 0 00 BSA 1000 a 0 00 BSA 225 E 0 00 E 0 00 Reset eE Import Export Run Log Contents A114 V7 Index Using the concentration you entered and the measured relative concentration the bioanalyzer software calculates a linear regression that can be displayed by selecting View gt Calibration Curve af Calibration Curve Re tate Cone agin Ea NOTE Calibration is only available for protein assays Changing the View of the Results A number of different options are available for viewing the data after it has been acquired by the Agilent 2100 bioanalyzer None of these options change the raw data but rather provide different means of viewing the results Overlaying Electropherograms Data from multiple wells can be overlaid within the single well large display view Hold down the CTRL key and then click the left mouse button on other lanes in the gel image in the lower left corner of the screen Sizing Standard A IgG reduced 200 150 100 Fluorescence sot Contents A116 V Index Bounding box
110. onnection Wizard 155 context sensitive help 161 163 copy functions 186 copying information 159 cross contamination 25 Cy5 labeled nucleic assay 192 Cy5 labeled nucleic acids data analysis 66 D data saving 138 data analysis 31 DNA 36 filtering parameters 36 protein 96 RNA changing 69 A 235 V Index RNA Cy5 66 data comparison program 194 data evaluation 35 128 136 accessing 54 DNA 36 protein 101 RNA 69 settings 36 data evaluation tool 128 alignment of electropherograms 133 loading electropherograms 130 peak find settings 136 sample number 132 data file reanalysing 62 data file reanalysing 124 data files limit number 132 naming 205 organizing 137 retrieving 137 data filtering 38 69 data handling 137 Data Organizer 152 data organizer 152 Data Organizer Connection Wizard 155 data points show 189 data points show hide tool 170 demo assays 191 description file 153 Contents diagnose instrument 194 DNA smear assays 49 DNA 7500 Smear 192 DNA assays 191 DNA samples 34 dsDNA 191 E edit samples 16 electrode cartridge 13 electrode cleaner 28 electrodes 228 electrokinetic forces 229 electropherogram DNA ladder 32 RNA ladder 67 electrophoresis 229 electrophoretic flow 229 end time 213 env file 153 error information 166 184 error messages 184 error occurred during the saving process 154 export auto export 210 export dialog 149 export file 153 exporting data 149 A 236 V Index F file extens
111. oosing the Global Peak Find tab X Demo DNA 7500 Properties xi Summary Ladder Analysis Min Peak Height 8 Reset Min Peak Width 0 5 Secs Slope Threshold 0 8 Sec Start Time 25 Secs End Time 95 Secs Filter Width 1 Secs Baseline Plateau 0 5 Secs Polynomial Order 6 IV Exclude Ladder use assay defined default values The Polynomial Order setting is used to define the power series applied to fit the raw data The higher the number you set the more the fit function will follow the noisy raw data curve As a result the noise level of the filtered curve will increase Contents A37V Index Filter Width defines the data window in seconds used for averaging The broader the filter width the more raw data points are used for averaging As a result the noise level will decrease but peaks will become lower and broader Overall changing the Filter Width has more effect on the result of the filtering procedure that is applied than does changing the Polynomial Order Peak Find Parameters After data filtering the Peak Find algorithm locates the peaks and calculates the local peak baselines The algorithm begins by finding all the peaks above the noise threshold in order to determine the baseline after which any peaks below the noise threshold are rejected A local baseline is calculated for each peak to allow for baseline drift Contents A 38 V Index The four peak find pa
112. ovide different means of viewing the results Overlaying Electropherograms Data from multiple wells can be overlaid within the single well large display view Hold down the CTRL key and then click the left mouse button on other lanes in the gel like image in the lower left corner of the screen l Ladder Ladder chicken liver MRNA chicken liver MRNA chicken liver MRNA Fluorescence A N 8 Y N 8 8 9 S a2 a7 52 57 62 67 72 Time seconds Contents A8V Index Bounding boxes will appear around the gel lanes signifying which wells are shown overlaid Each peak graph will be shown in a different color and line style with a legend at the top of the window You can remove wells from the overlay by CTRL clicking the corresponding lane in the small gel display the bounding box will disappear 1 willl L1i2345678 9 0 12 Only ladder selected Ladder with samples 1 to 3 overlaid Contents A 86 V Index A stand alone program called Data Evaluation is also included with the bioanalyzer software and can be accessed by choosing Tools gt Compare Results This program allows you to compare the results from the same or different runs and even different assays within a single window and provides tools that allow you to manipulate the comparison of the data in different ways Documentation and help for the Data Evaluation program are available within that program Contents A 87 V Index Changi
113. r choice provides more detail 1 280 colors since it maps the signal into a larger color space than is available with the other monochrome options 256 levels of brightness View E Single Well F2 All Wells F3 F4 E View Get Combined Results 4 Previous Well gt Next Well J E Black on White Standard Curve White on Black Cabin Cive Sl REPRE T Chip Run Summary Green on Black Fa Magenta on White Gl Red on Black E Pseudo on black Contents A5 V Index Force Baseline to Zero Since all electropherograms show some amount of background fluorescence the bioanalyzer software automatically sets the baseline to zero fluorescence units To remove the zeroing select Tools gt Options gt Advanced and uncheck the Zero Baseline box 450 400 350 Zero Baseline 300 250 200 Fluorescence 150 100 5 he j f E 1 1 1 1 a a n f j 1 j j 1 T 1 1 l T T 25 30 35 40 45 50 55 60 65 70 75 80 85 30 Time seconds 550 500 40 Non Zero Baseline 400 350 300 Fluorescence 250 200 150 100 a _ 4 T T r i 25 30 35 40 45 Contents 1 f if 1 T T i T I T 50 55 60 65 70 75 80 Time seconds 45y Index Baseline Correction Ladder The individual sample settings tab for the ladder well in a DNA assay shows a check box for Baseline Correction enabled by default In case of bend ladder baselines the baseline correct
114. r markers DNA 34 lower marker 44 Lower Marker Present 214 M main screen 20 manual alignment 134 marker peaks 34 aligning or unaligning 46 80 assigning 44 sample 46 measurement practices 25 menu bar 166 167 menus 185 microchannels 228 microfluidics 230 Mig Time results table 61 migration time 31 DNA 33 Min Peak Height 40 Min Peak Width 40 Molarity nM results table 61 molecular separation techniques 230 molecular sieving 228 A 238 V Index mouse notations in help 164 mouse shortcuts 225 mRNA Smear 192 mRNA Smear Nano 192 multi instrument system 21 noise level 37 noise threshold 38 0 open data file dialog box 202 Open icon 168 Options advanced tab 209 chip alert tab 208 data files tab 205 options reader tab 207 options dialog box 195 205 organizing data files 137 overlaying well graphs 52 85 P parts 231 peak find parameters 104 peak find algorithm 31 38 RNA 66 peak find parameters 38 Contents RNA 71 peak find settings data evaluation tool 136 peak find settings global 212 peak Info 189 peak number results table 61 peak excluding a 45 peaks finding 40 physical specifications 165 pipette 25 pipette tips 25 polynomial order 37 214 principles nucleic acid sizing 228 Print 140 print auto print 210 setup 147 print dialog box 218 printer driver 147 Print Icon 169 printing 137 4 wells per page 146 assay summary report 142 electropherogram 144 gel report 143 help 162 repo
115. r more information You can also save files manually by choosing File gt Save or File gt Save As NOTE After data has been acquired and you make changes to the file display the raw data acquired from the Agilent 2100 Bioanalyzer is not changed only the display of the data is changed and saved If you alter the data shown in any way after it has been saved and try to exit the software or acquire new data a dialog box will appear asking whether or not you wish to save the changed file The Save As dialog box contains a check box for saving the file as read only A read only file may be opened the title bar will show Read Only at the end of the filename and edited but may not be saved under the same filename If you attempt to save an edited read only file and error message will be displayed explaining that the file is a read only file Clicking OK in the error box will open the Save As dialog box Entering the same name as the read only file causes another error message to be displayed telling you to save the file with a different name or to a different location The benefit of saving a file as read only is to prohibit you or other users from making changes that would alter the file in any way Since the Agilent 2100 Bioanalyzer software allows you to open data files reanalyze them using different assays alter peak find parameters etc and saves these new parameters with the file when it is saved you may prefer to ensure that t
116. r the list click Remove all Files if Upload to Data Organizer AddSelectin SelectAll 3 Invert Selection 1 RemoveSelectedFiles X lt Remove allies gly Start Batch Upload o Agilent 2100 Bioanalyzer 5 Bio Sizing E Assays E Configuration Data a Packets BioSizina DNA 1000_00000_2001 08 08_08 02 17 cid 2 BioSizing_DNA 1000_00000_2001 08 08_08 02 17 log BioSizina DNA 10 2001 08 09 01 52 03 cld File Path C Program Files Agilent 2100 Bioanalyzer Bio Sizing Data Bio g C Program Files Agilent 2100 Bioanalyzer Bio Sizing Data Bio C Program Files Agilent 2100 Bioanalyzer Bio Sizing D sta Bio Ef C Program Files Agilent 2100 Bioanalyzer Bio Sizing Data Bio Ef C Program Files Agilent 2100 Bioanalyzer Bio Sizing D sta Bio Ef C Program Fles Agilent 2100 Bioanalyzer Bio Sizing D ata Bio CAProgram Files Agient 2100 Bioanalyz C Program Files Agilent 2100 Bioanalyz C Program Files Agilent 2100 Bioanalyz C Program Files Agilent 2100 Bioanalyz C Program Files ailent 2100 Bioanalyz C Program Files Agilent 2100 Bioanalyz 2 BioSizina_DNA 1000_00000_2001 08 09_01 52 03 log BioSizing_ONA 1000_00000_2001 08 10_05 58 32 cld 2 BioSizing_DNA 1000_00000_2001 08 10_05 58 32 log BioSizing_DNA 1000_00000_2001 08 13_05 08 29 cld 2 BioSizing_DNA 1000_00000_2001 0813_05 0823 eg s BiaSizinn DNAANNN NONN INM NA R 06 20 72 old rH jal pe a ee E stb 6 File s 1 selected Bi
117. rameters that can be changed Min Peak Height Slope Threshold Min Peak Width and Baseline Plateau are highlighted and explained in the table below Choosing OK sets the parameters for all the wells X Demo DNA 7500 Properties x Summary Ladder Analysis Min Peak Height 8 Reset Min Peak Width 0 5 Secs Slope Threshold 0 8 Sec Start Time 25 Secs End Time 95 Secs Filter Width 1 Secs Baseline Plateau 0 5 Secs Polynomial Order 6 VV Exclude Ladder use assay defined default values The Reset button sets the Global Peak Find values back to the factory settings Contents A39 V Index Min Peak Height Slope Threshold Min Peak Width Baseline Plateau Exclude Ladder Determines the threshold for the peak find algorithm For each peak the difference between the start point value and the center point value local baseline must be greater than the Minimum Peak Height value Determines the difference in the slope that must occur in order for a peak to be recognized The inverse of this value is used to determine the peak end Determines the minimum amount of time that must have elapsed after he threshold was exceeded A parameter that assists in finding peaks The signal is recognized to be at baseline whenever the slope of the data is less than the Slope Threshold setting either positive or negative for longer than the time set for the Baseline Pl
118. red across invocations of the application NOTE As a supplement to the bioanalyzer the Agilent 2100 Bioanalyzer Data Organizer provides a central repository for the digital data generated by the 2100 Bioanalyzer system software 2100 Bioanalyzer chip data files can be uploaded to the data organizer server allowing for shared access and parameterized searches see Uploading Chip Data to the Data Organizer on page 152 for more information Contents A18V Index For more information see Data Analysis DNA and DNA Smear Assays 31 Data Analysis RNA Cy5 Labeled Nucleic Acids and RNA Smear Assays 66 e Data Analysis Protein 96 Further tasks include Changing Your Data Analysis DNA and DNA Smear Assays 36 Changing Your Data Analysis RNA and RNA Smear Assays 69 Changing Your Data Analysis Protein 101 You can print a report Printing an Assay Summary Report 142 Printing a Gel Report 143 Printing an Electropherogram 1 44 Printing 4 wells per page Electropherogram 146 Or you can export data Exporting Data 149 Uploading Chip Data to the Data Organizer 152 Contents A19 V Index Starting the Agilent 2100 Bioanalyzer Software Single Instrument System To start the software go to your desktop and double click the icon E The main screen of the program appears The Agilent 2100 Bioanalyzer or a chip should be represented at the left side of the screen what is sho
119. rotein chips Improper vortexing can ead to poor results Do not force the chip into the receptacle of the Agilent 2100 Bioanalyzer Proper placement of he chip should not require force Improper placement of the chip could damage the electrode assembly when you close the lid Check whether the chip selector is in the correct position Do not touch wells of the chip The chip could become contaminated leading to poor measurement results Do not leave any wells of the chip empty or the assay will not run properly Add 1 pL of sample buffer to each unused sample well For proteins add 6 pl of sample or ladder replicate in each empty sample well Do not touch the underside of the chip Contents A271 V Index Agilent 2100 Bioanalyzer Do not touch the Agilent 2100 Bioanalyzer during a run and never place it on a vibrating surface Clean electrodes daily by using the electrode cleaner ona quarterly basis by using a toothbrush and distilled water For more details see Maintenance and Troubleshooting Guide Electrode Cartridge Maintenance Clean the focusing lens on a quarterly basis or after any liquid spill using isopropanol For more details see Maintenance and Troubleshooting Guide Lens Maintenance Contents A 28 V Index Decontamination Procedure for RNA Assays Perform the following decontamination cleaning procedure on a daily basis before running any RNA assays See Maintenance and Troubleshooting Guide
120. rt 133 80 Secs pes End 34 90 Secs Color Bi Cancel Apply the RNA alignment and the Baseline properties The RNA tab is available only for the RNA assays It allows to add and delete individual peaks that you identify manually Contents A178 V Index Results tab and Error tab give you information on results and possible errors Region Tab Sample Settings ANA Results Enors Region Region i Add From fi 000 bp r To oo be Delete Color M Reset Sancel Bhange The Region tab is available only for smear assays It allows definition of certain regions of interest defined in terms of size bp which allows characterization of broad bands smears The software determines the size of each region in relation to the total area of the smear in percentage On the Region tab you can add delete and reset regions Each region is indicated by two colored lines Contents A179 V Index Large Gel View logios 2100 Bioanalyzor Bio Sizing BioSizing DNA 12000_00000_2001 07 05 16 09 22 fi Graph Asoay Toole Help H 8 DOA B oa amp amp mar Jale Kara rue siosizng_oma 1200_ 0o00 _2001 0 05_16 19 32 Assay vemo ona 1200 J sei Paaa onnyeuRe sepa Bs Laddi NEST kb Ad2 Dra Ad2 BgIl Adz Pval High Mass Sunihetc NEB THE Ad2 Dial AGz BaIIl Ad2 Pvu I Hgh Mass Synthetic Teder laide DNA badder Badde TINA 170 ms 10380 m _ 7000 S000 3000 2000
121. rt 140 protein assay 192 A 239 V Index purified proteins 192 R RAM 132 raw data 31 38 read only 202 reagent kits 232 reagent mixes 26 reagents 26 232 reanalyzing a data file 92 Region Tab 179 report printing 140 report file 153 result table 45 copy 186 copying 160 exporting 150 result tables exporting 149 results table 61 protein 123 RNA 91 view 176 results view changing 52 ribosomal RNA 68 RNA 191 alignment 73 data analysis 66 Contents smear assays 82 rRNA bands 76 ratio 77 run log 195 view 173 S sample information 199 samples edit 16 samples DNA 34 Save Data As dialog box 219 Save Icon 168 saving selected wells 220 scale icon 169 scaling 189 sensitivity 136 serial port 207 settings Connection Wizard 155 settings saved with assay 215 shortcuts 221 signal to noise ratios 136 single well display options 177 single well view 175 176 183 Single Well view icon 169 Size bp results table 61 A240 V Index slider 56 slope threshold 40 136 213 small display 166 small gel view 182 183 small graph view 183 smear assays DNA 49 RNA 82 software 231 software algorithm 31 software reference 166 spare parts 233 specifications 165 start button 15 start dialog box 15 193 196 start time 213 status indicator 13 status line 166 184 storage limit 209 study information 198 system log 195 T temperature monitor 194 time window for analysis 43 Time parameters 43 tips
122. rtain trend in the migration time for example a smiling curve 3 Find the first lane with the incorrect marker setting and click on that lane 4 Set the marker by right clicking on the peak that is the correct marker peak and choosing Manually Select Lower or Upper Marker 5 Repeat this procedure for all lanes with incorrect marker settings 6 Switch Analysis back on by using the Turn Off On Analysis icon in the toolbar Contents A488 V Index Analyzing DNA Smear Assays DNA smear assays are designed to analyze broad bands as they occur with fragmented genomic DNA or double stranded cDNA These assays allow you to define regions in terms of base pairs that elp you to characterize dsDNA smears You can add delete and change regions on the Region tab on the right of the result table in the single well view Sample Settings Region Results Enrors Region 2 Add From fioco bp Delete To 2000 bp Reset Color aml Apply The total area to be evaluated is determined by two dashed lines the lower and upper time markers These time markers are displayed between the lower and upper DNA markers of the sample You can move the lines with the mouse to get a horizontal baseline But if you change the total area time markers the total area will change and also the result which is displayed as of the total area To add a new region 1 Click the Add button 2 Enter the From and To parameters for the region
123. s the global peak find settings to all wells Choosing No allows individual wells to retain changed peak find settings NOTE This prompt appears whenever at least one of the samples has different local settings Contents ATV Index Manually Moving Start and End Points of rRNA Bands It is possible to manually alter the start and end points of the rRNA bands in an RNA or Cy5 labeled nucleic acids assay Zooming in on the base of a particular peak allows you to see the start and end points color coded to match the designator shown on the RNA tab of the sample information pane Positioning the cursor over one of these points changes the cursor to a pointing hand allowing you to click and drag the point along the line of the peak until it is positioned as desired R Vew Giarh Assay Tods Heb 6H SHO B B t EAA Fek Ho E Dot File BioSizing ToteLANA_oooeD 2002 02 24 42 42 06 Masay Domo Eukaryote Total RNA ey Sises Eset eee amp ee ey Tes i rouse seen RNA 3 l i 2 i ena taba at i i i Bis tl g Fl FI zt i et i J SHl 5 ca asi L F i Ty iyi i t t t t t t 1 a 2a aa 30 a sa 3 Time seconds z Pek aaa Tonies Ganplo Seting ANAL Reute Enor PAT a winPeakHecht 05 Goa 3 aw oer vinPeacwiah 03 pow i as cer a Sone heshad 07 E FI n87 Te 2S Bassie StatTine 20 00 Secs 7 44 80 61 53 EndTime 59 00 Se
124. s the status Lid open i Lid closed but no Dimmed icon instrument switched off chip inserted or not communicating properly see Maintenance and Troubleshooting Guide Contents 41y Index Use the assay run previously the default assay showing or choose a new one from the Assay menu Assay dsDNA BNA Protein Other Smear Demos Demo DNA 7500 Properties yvrrYYY Lt Save Assay As DNA 101 DNA 7500 Eukaryote Total RNA Nano mRNA Nano Prokaryote Total RNA Nano Start 4 Prepare the buffers samples and chip Protein 200 Protein 200 Plus Cy5 Labeled Nucleic Acids Cy5 Labeled Nucleic Acids Nano Eukaryote Total RNA mRNA Nano Prokaryote Total RNA DNA 12000 Smear DNA 7500 Smear mRNA Smear mRNA Smear Nano For more information see the appropriate Reagent Kit Guide 5 Place the chip in the Agilent 2100 Bioanalyzer and close the lid Contents Index The following figure shows the major components of the Agilent 2100 Bioanalyzer Lid Electrode Cartridge Chip Selector Base Plate Lens Status Indicator For more details about the hardware and instructions for exchanging the electrode cartridge please refer to the Maintenance and Troubleshooting Guide Contents A113 V Index The lid contains a slot which accommodates an electrode cartridge The cartridge contains 16 electrodes that fit into the wells of the
125. t 2100 Bioanalyzer Choices are to turn the sound off leave it at the default sound setting or use a custom sound which can be any wav file of your choice You can also change the interval in seconds between the alert sounds from the default at 1 second to a maximum of 15 seconds Contents A 208 V Index Advanced Tab Data Files Reader Chip Alert M Zero Baseline T Gel Mobility Correction I Limit the storage of raw data backups to a total of 50 MB of disk space T Auto Print Settings I Auto Export Settings OK Cancel Zero Baseline The Zero Baseline setting is used to offset the graphs shown for the individual wells but does not affect analysis Limit the storage You can also choose to limit the storage of raw data backups to a certain amount of disk space The default is 50 MB which corresponds to 50 assays When the limit is reached the data of the first assay is overwritten Contents A 209 V Index Gel Mobility Correction Auto Print Auto Export Gel Mobility Correction is applied to the gel display Each point is plotted against mobility instead of time where mobility equals distance time By plotting against the reciprocal of time 1 time the separation of the peaks is proportional to the mobility and is more comparable to a photo of a real gel Applying mobility correction expands the fast peaks and compresses the slower ones Enable the check box to activate the Auto Print mode Ever
126. t assays even for the same type of molecules for example between DNA 1000 and DNA 7500 assays Make sure you are familiar with the Essential Measurement Practices 25 Prepare the reagents load the chip and run the assay as described in the appropriate Reagent Kit Guide Contents A30V Index Data Analysis DNA and DNA Smear Assays How the Agilent 2100 Bioanalyzer Software Analyzes Data The purpose of bio sizing assays is to calculate the size and or concentration of nucleic acid fragments Results are calculated after all data for an individual well has been read The data analysis procedure consists of the following steps 1 Raw data is read and stored by the system for all the individual wells 2 A software algorithm filters the data and plots the resulting electropherograms of all wells You can change the settings of the filtering algorithm after the run and reanalyze your data 3 Peaks are identified for all wells and tabulated by migration time You can change the settings of the peak find algorithm after the run has finished and reanalyze your data Contents A31V Index 4 ADNA ladder a mixture of DNA fragments of different sizes is run first from the ladder well see the electropherogram below The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed Ladder 120 110 100 90 80 4 trhrrrhrrrhrrhrrrhrrh 70 60 50 4 Fluorescence 4
127. ta curve As a result the noise level of the filtered curve will increase Enables RNA alignment to all wells of the sample for RNA assays only Subtracts the lower marker s part of the total area Lower markers are only added for RNA assays with the suffix nano Excludes the ladder from any changes you may make to the peak find settings the default ladder settings from the assay are used instead Enables the baseline correction to get a horizontal baseline Enables the calibration to an added standard protein only protein assays If you save the changes you make the new peak find settings are saved along with the file and will be used the next time that file is opened Clicking the Reset button causes the Peak Find settings to revert to the values saved previously Contents A214 V7 Index The Peak Find tab has an additional Save As button Clicking this button allows you to save the current values entered for the Peak Find settings as the defaults for a new assay or to save the current values with an existing assay The default assay folder will be shown but you can save the assay to any folder of your choice NOTE Settings that are saved with an assay file are the peak find settings gel color default and well names Contents A215 V Index Combined Results View Dialog Box X Combined Result View xi Well Range C AllWells lg Wells i 3 5 Enter well number and or well ranges separated by comm
128. tal RNA Nano mRNA Nano Prokaryote Total RNA Nano A 190 V Protein 200 Protein 200 Plus Cy5 Labeled Nucleic Acids Cy5 Labeled Nucleic Acids Nano Eukaryote Total RNA mRNA Nano Prokaryote Total RNA DNA 12000 Smear DNA 7500 Smear mRNA Smear mRNA Smear Nano Index The Assay menu includes the default assays that are shipped with the Agilent 2100 Bioanalyzer software as well as any assays that have been created and saved subsequently into the Assays gt Assays type folder dsDNA The dsDNA assays included in the Agilent 2100 Bioanalyzer DNA 12000 software are four Demo assays which can be used as a DNA 7500 learning tool and four assays designed for separation of DNA DNA 500 To choose an assay that is different from the one run previously DNA 1000 the default for which Properties are shown in this menu choose Assay gt DNA gt one of the assays from the pull down menu t is possible to modify the parameters for one of the assays shipped with the Agilent 2100 Bioanalyzer software and save it as a new assay for future use RNA The RNA assay is designed to separate RNA up to 6000 base Eukaryote TotalIRNA Nano pairs Different RNA assay scripts included with the Agilent mRNA Nano 2100 Bioanalyzer software are designed to determine the Prokaryote TotalRNA Nano concentration of total RNA or mRNA preparations Nano assays Contents contain a lower marker A191 V7 Index Other Cy5 Labeled Nucleic Aci
129. the bioanalyzer software automatically sets the baseline to zero fluorescence units To remove the zeroing select Tools gt Options gt Advanced and disable the Zero Baseline box Baseline Correction Ladder and Samples The individual sample settings tab for the ladder and sample wells in a protein assay shows a check box for Baseline Correction enabled by default In case of a drifting baseline baseline correction can be used to flatten the baseline To enable the baseline correction select the Baseline Correction check box and click Apply Sample Settings Results Enors Min Peak Height 2 0 Min Peak Width 0 2 Slope Threshold 40 I Baseline Correction Contents A121 V7 Index HSA 150 250 Baseline correction disabled 200 g 150 5 100 i 50 a o o mno s o ok a l oT 25 30 35 40 45 Time seconds T HSA 150 200 175 Baseline correction enabled 100 75 Fluorescence 50 235 Time seconds Contents A12 Y Index The Results Table The Results Table appears below the single well view in the large display area This table provides the following information Peak Number Mig Time seconds Corr Area Size kDa Relative Conc pg mL Calb Conc pg ml The order in which the peaks were detected The m
130. thm locates the peaks and calculates the local peak baselines The algorithm begins by finding all the peaks above the noise threshold in order to determine the baseline after which any peaks below the noise threshold are rejected A local baseline is calculated for each peak to compensate for baseline drift Contents A 103 V Index The four peak find parameters that can be changed Min Peak Height Slope Threshold Min Peak Width and Baseline Plateau are shown below Protein 200 Plus Properties xi Summary Ladder Analysis Min Peak Height 2 Min Peak Width Secs 0 2 Reset Slope Threshold Sec 4 Start Time Secs 15 End Time Secs 47 Filter Width Secs 0 5 Baseline Plateau Secs 0 3 Polynomial Order 6 I Calibrate all Proteins M Baseline Correction IV Exclude Ladder keep current values Save As OK Cancel Leaves K The Reset button sets the Global Peak Find values back to the factory settings Contents A104 V7 Index Min Peak Height Slope Threshold Min Peak Width Baseline Plateau Exclude Ladder Baseline Correction Calibrate all Proteins Determines the threshold for the peak find algorithm For each peak the difference between the start point value and the center point value local baseline must be greater than the Minimum Peak Height value Determines the difference in the slope that must occur in order for a peak to begin The
131. tive window is highlighted and any other window title bars on your desktop are dimmed Dragging the title bar repositions the window on the screen in window view only if the window has been maximized dragging will not work The buttons that appear at the right end of the title bar can be used to minimize the window so that it appears only on the task bar maximize the window to full screen size or to close the window Menu Bar File Edit View Graph Assay Tools Help The menu bar is the area across the top of the window directly below the title bar It contains the names of the menus that group together related commands Clicking a menu name displays a list of commands that can be used to access software functions Contents A 167 V Index The menus contained in the Agilent 2100 Bioanalyzer software menu bar are File Menu 185 Edit Menu 186 View Menu 187 Graph Menu 189 Assay Menu 190 Tools Menu 194 Help Menu 196 Tool Bar S H 6 0 O B A amp B Pesk Nube Jal gt Each button on the tool bar represents a menu command and is a shortcut to activating that command The buttons on the tool bar are Open Brings up a dialog box allowing you to open a previously saved data file Save Saves the data file currently showing on the screen AD t Contents A 168 V Index lt E Ee i tak red Contents Print Opens a dialog box asking you to choose the elements you wou
132. trument button The set application can be changed by a double click Contents A23V Index Closing the Launcher To close the Launcher first you must close all open instances of the Agilent 2100 bioanalyzer software Then position the mouse cursor over the Launcher icon and click the right mouse button The following menu will appear Set Application Change Serial Port Setting Hide Title Bar Minimize Stay On Top Remove Shortcut from Start Menu Minimize All Help About Close Select Close to terminate the Launcher NOTE You can change the port settings of the different Agilent 2100 bioanalyzer instances by choosing Change Serial Port Setting Refer to the online help for more details Contents A24V Index Essential Measurement Practices This section lists all relevant hints regarding the handling of tools chips reagents and the Agilent 2100 Bioanalyzer For the latest information on assay related hints go to the Lab on a Chip web site at http www agilent com chem labonachip Tools and Handling Always wear gloves when handling chips to prevent them from becoming contaminated e When pipetting sample use pipette tips that are small enough Pipette tips that are too large will lead to poor quantitation accuracy e Change pipette tips between two pipetting steps to avoid cross contamination Always insert the pipette tip to the bottom of the well when dispensing the liquid Placing
133. uide in the Help menu Contents A6V Index Contents Welcome sisstescscascnsccatstcsscesdcasccsccusensctosctscuascsetnctunatssecuer stvecestsevatancseoscostastintect uedtaceversnieecectsneds 3 What s New How to Use This Guide Navigating within Acrobat Reader ccccccscessessssssecesessssssssseesssesesssesseesesseeseeseesecstsaseneeses 6 Quick Step Overview sisississicccccccssiscsccsscissiessciteiscaesncceiaiosveasctesscvene scsdeissveassvardssonsacavcatevensacoiss 11 Starting the Agilent 2100 Bioanalyzer Software Single Instrument System 20 Starting the Agilent 2100 Bioanalyzer Software Multi Instrument System 21 Essential Measurement Practices scsssssscsssssessenssssseseeeeseesesesteesneeseeesessteensneeseneneenes 25 Tools and Handling Reagents and Reagent Mixes General Gel and Gel Dye ooo eeeececeececccsceesesssesessesssssscssessssussscsscsessssssesecsssuesnseusesssnesusesssnsaucensssssussuseseseensens 26 Decontamination Procedure for RNA Assays Preparing and Running an ASsa csssscsssssssssesessesessesssessesesssneesseeaeesaesseatsnseseesaees 30 Contents ATV Index Data Analysis DNA and DNA Smear AssaJS scsssssssssesssseesssseesseseseseeesesseneeeerseaes 31 How the Agilent 2100 Bioanalyzer Software Analyzes Data c cccccescesssssessessestesessnesteeeeees 31 Changing Your Data Analysis DNA and DNA Smear Assays
134. ular Molecular comi mM coma M coms M coms m 4 Click on the first instrument button to start an instance of the software It will establish communication to the instrument connected to COM port 1 By clicking on the second third and or fourth instrument buttons you can start new instances of the software establishing a connection to the instruments attached to those ports NOTE You can communicate with a maximum of four instruments using the Launcher Contents A22V Index 5 After having established communication to the first instrument the Launcher will look as shown below Agilent Technologies Bioanalyzer Launcher Molecular Molecular Molecular Molecular comi coma F coms M coms M The Launcher can be described best by dividing it into the following sections Instrument Buttons Four boxes that represent the instances of the bioanalyzer software Beneath each button is the status of the connection or Demo Mode COM Ports Four COM port designations followed by LED representations showing the port to which the instrument associated with that instance of the bioanalyzer software is connected that instrument s status and the selected application Arrange Buttons On the right two buttons allow you to display the instances of the program as either tiled or cascaded on the computer monitor Application Buttons Indicate which software application can be started by double clicking the corresponding ins
135. ults Enors Min Peak Height 05 Global Min Peak Width 05 Baw Slope Threshold 0 8 Baseline Start Time 22 00 Secs End Time 69 00 Secs ent Cancel You can set a lower or upper marker manually To select a marker use the context menu that is available by right clicking on one of the bands of the sample All wells with enabled RNA alignment are actualized Contents A81V Index Analyzing RNA Smear Assays RNA smear assays are designed to analyze broad bands as they occur with mRNA cDNA and cRNA These assays allow you to define regions in terms of base pairs that help you to characterize the smears You can add delete and change regions on the Region tab the part of the stack on the right of the result table Sample Settings Region RNA Results Enors Region 1 Add From 850 Delete To 3500 b Color il Reset Cancel Apply The total area is determined by two dashed lines the lower and upper time marker You can move the lines with the mouse to get a horizontal baseline To edit the lower and upper time marker you have to click the Settings tab to the front In this view you can move the time marker by using the mouse The total area that is defined in this view is also used for calculating the value of the region If you change the total area time markers the total area will change and so the result which is displayed as of the total area To add a new region 1 Cli
136. un with samples or use of internal assay ladder Assay you can save the changed settings under a new assay name if desired NOTE If you save the data file after making changes it will keep a record of the assay if a new assay name has been saved it will use the settings from this assay the next time the file is opened gel color well names and peak find settings that were in effect at the time the file is resaved If you do not want to change the original file choose Save As and give the file a new name or save it to a different location Combining Results If you want to combine the results of different wells you can select these and then print a table view of the results To do this 1 Click View gt Combined Results 2 Select the wells to be combined 3 Click OK to display the combined results in a table view The items that can be changed for combining results Well range All Wells to combine the results of all measured wells e Wells to combine the results of selected wells Contents A 63 V Index Options Exclude Markers to display the values without the markers Include Ladder to display the values of the ladder in a separate table Combined Result View 5 Contents At4YV Index The results are displayed in a tabular format ed Result View BEE BioSizing_DNA 1000_00000_2001 07 03_13 26 45 cld
137. ware provides a central repository for the digital data generated by the 2100 Bioanalyzer system software If the Data Organizer Client is installed data can be uploaded directly from within the bioanalyzer software Contents A4yvV Index How to Use This Guide Use the interactive bookmarks in this frame to choose your desired topic Use Acrobat Reader s navigation bar to move around within a topic see Navigating within Acrobat Reader on page 6 Acrobat Reader Users Guide_pdf File Edit View Tools Window Help ld gt gt i gt Dan aj L Welcome gt O How to Use This Guide O Contents O Quick Step Overview O Starting the Software lt D Preparing and Running an Assay D Reagent kits C Procedures Click here to go to the table di contents Click here to go to the index Here s the current page number A displays previous page V displays next page Contents A5V Index Navigating within Acrobat Reader When you ve chosen a topic with the bookmarks use the buttons in Acrobat Reader s tool bar to move around within a topic Returns to the previous view Click several times to redo more view changes s Returns to the next view Click several times to redo more view changes Displays the next page Displays the previous page Displays the first page Displays the last page For more information see the Acrobat Reader Online G
138. wn depends on the status Lid closed no chip or chip empty Fe ER Vion Graph Asoy s kW amp DOBE 2 ala ay se aes FOR et POR M2 POR Mina Lid open F Dimmed icon no communication Lid closed chip ou EE sE inserted DNA or demo selected Lid closed chip inserted RNA or demo selected FOR Mit FoR FoR Lid closed chip inserted Protein or demo selected Read Die Deno Node faa Exo aah 7 Contents A2V Index Starting the Agilent 2100 Bioanalyzer Software Multi Instrument System 1 To start the multi instrument software you must start the Agilent 2100 Bio Sizing Launcher You can start the Launcher by choosing Start gt Agilent 2100 Bioanalyzer gt Utilities gt Bio Sizing Launcher rograms Ly fE Agilent 2100 Bioanalyzer Bio Sizing Help Ef Bio Sizing JB Bio Sizing Installation Qualification Data Evaluation X Bioanalyzer Launcher Ri ReadMe NOTE You cannot start the Launcher when the Agilent 2100 Bioanalyzer software is already open Contents A21V Index 2 Upon starting the Launcher this way for the first time a message will appear asking if you would like the Launcher to start automatically each time Windows is started X Bio Sizing Launcher Would you like to configure the Bio Sizing Launcher to start automatically whenever Windows is started Agilent Technologies Bioanalyzer Launcher Molecular Molecular Molec
139. y new data file generated will trigger a print out Clicking the Settings button brings up the Print window It allows to define the type of print outs To learn more about printing see Printing a Report on page 140 Enable the check box to activate the Auto Export mode Every new data file will be exported Clicking the Settings button brings up the Export window It allows you to select the type of exported data files To learn more about exporting data see Exporting Data on page 149 Contents A210 V Index Assay Properties Dialog Box The Assay Properties dialog box displays the settings used to determine the ladder peaks and other values required for analysis The settings in this box with the exception of the settings on the Peak Find tab are not changeable by the user emo DNA 7500 Smear Properties E Ladder Analysis Global Peak Find Regions Name Demo DNA 7500 Smear Tile DNA 7500 Smear Analysis Demo Comments Copyright 2001 Caliper Technologies Corp 1 0 Initial Release Keywords Demo SimDNA7500Smear txt 1 125 Location C Programme Demo DNA 7500 Smear asy Version f E Format 1 16 0 Save As OK Cancel Contents A211 V7 Index Global Peak Find Settings Choosing Assay gt Assay Properties opens the Assay Properties dialog box The settings found on the Global Peak Find tab can be changed to alter the way in which the program locates peaks or fragments the other tab settings ar
140. ze the data after the run has finished Note that peak find settings can be changed either for all or only for certain wells Contents A 96 V Index 4 Asizing ladder see the example electropherogram below which is a mixture of proteins of different molecular weights is run first from the ladder well The sizes of individual proteins in kDa are preset in the assay and cannot be changed Fluorescence 5 A standard curve of migration time versus size is plotted from the sizing ladder by interpolation between the individual protein size migration points The standard curve derived from the data of the ladder well should resemble the one shown below If not please refer to the Maintenance and Troubleshooting Guide Standard Curve Of x Point to Point Fit raj Cuve FII Lader Fremeni m 2 m a g i E 10 a D 15 a z E a s Time ecords Contents AIS V Index 6 Two proteins upper and lower marker are run with each of the samples as an internal standard bracketing the sizing range The lower marker peak is followed by a system peak The lower marker and upper marker are internal standards used to align the ladder data with data from the sample wells The figure below shows an example of assigned marker peaks in a sample well 1104 Lower System Peak 1004 Marker 90 80 4 704 60 4 504 404 Fluorescence 304 20 104 o 1 TTT a

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