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KCjunior Users Guide - Bio-Tek

1. 7 Optional Manually assign a Plate ID to this plate gt For multiple plate endpoint absorbance protocols each individual plate can have its own Plate ID assigned This prompt appears before each plate is read 8 Place the plate on the reader then click OK to begin the plate read gt If the attached reader is equipped with a barcode scanner and Use Reader s Barcode Scanner for Plate ID is enabled in KCjunior under Setup Configuration General KCjunior attempts to scan the plate If no barcode is detected or if the attached reader is not equipped with a barcode scanner the Plate ID can be entered manually gt Click the Abort Read button to halt the read in progress Data reduction is based on any data retrieved up to the time the button was clicked gt When the plate read is complete KCjunior automatically retrieves the measurements from the reader performs data reduction and displays the results See Viewing Results or Analyzing and Manipulating Results for more information 9 To save the results gt Select Results Save Results or press CTRL S KCjunior requests a Results ID if one was not defined before the plate read Enter the ID using up to 32 alphanumeric characters This is the name under which the record will be stored in the Results Database Click OK to save the results User s Guide to KCjunior Reading Plates e 6 11 Note Simulation Data is not supported for the spectral scan read method
2. Comments and Warnings in the Troubleshooting section of this guide for more information Automatic Sensitivity Adjustment Process The automatic sensitivity adjustment process for the Scale to Low High Range option is performed as follows Using the filter set parameters provided an initial reading is performed on the designated pre reading wells using a sensitivity setting of 25 A signal to noise ratio is calculated If it is found to be valid the sensitivity setting and the value are retained for further evaluation The sensitivity is then increased by increments of 25 and additional readings are made until one of the following conditions are met Condition 1 Success If two successive valid signal to noise ratios are within either 0 1 or 5 percent of each other then 2 additional readings are performed using sensitivity settings at one quarter increments between the sensitivity of those readings For example if the signal to noise ratio at sensitivities 50 and 75 are 120 31 and 120 38 respectively then additional readings will be performed at sensitivities 55 60 65 and 70 The valid signal to noise ratios of the 6 successive readings are then compared to determine the highest value The sensitivity of the highest signal to noise ratio is then chosen for the actual filter set read If the highest signal to noise ratio occurs at multiple sensitivity settings then the highest sensitivity will be selected
3. Read Plate Simulation Data 6 12 e Reading Plates Simulating a Plate Read KCjunior provides the option to simulate a plate read by entering plate data manually or by copying data from a spreadsheet and pasting it into a KCjunior plate Simulation data typically represents previously acquired measurements entered into KCjunior for analysis Simulation data can be generated with or without a protocol open and it can be saved accessed and modified To simulate a plate read 1 Ensure that under Setup Reader the desired Reader Type is currently selected The reader does not need to be connected to the computer to simulate a plate read 2 If desired open an existing protocol or define a new one Initiate the simulation by clicking the Read Plate button in the KCjunior main screen selecting Results Read Plate or by clicking the toolbar icon The Read Plate dialog appears 4 Enter a Results ID using up to 32 alphanumeric characters The ID is optional in this dialog but it will be required later if the results are to be stored in the Results Database gt If performing a multiple plate endpoint read all of the plates in the read will be stored in the Results Database under this Results ID 5 Enter a Plate Description using up to 1024 alphanumeric characters This description is optional 6 Click the Simulation Data button Note If there is no protocol open the Read Method dialog appears Enter the basic
4. Use brackets to reference a well by its location on the plate For example e A2 A12 2 A12 e X C2 C3 Use a dollar sign to retain the row and or column reference when copying formulas from cell to cell in the template e Al A1 A 1 and A 1 all refer to well Al e A1 copied from Al to B1 becomes B1 e A1 copied from Al to B2 becomes A2 e A 1 copied from Al to B2 becomes B 1 User s Guide to KCjunior X The letter X represents the value of the current well e X BLK in well G6 subtracts the BLK mean from the value in well G6 e X A2 in well Al subtracts the value in well A2 from the value in well Al e Xis not valid within a Cutoff formula lt well id gt Any Well IDentifier used in a formula represents the group mean or average e PC represents the mean of all PC wells on the plate e SMPO01 represents the mean of all SMPOI wells on the plate e SD BLK calculates the standard deviation of the BLK wells Order of Precedence KCjunior processes formulas according to a certain order of precedence 1 parenthesis 2 unary minus 3 and 4 and e Operators and are of equal precedence e Operators and are of equal precedence e Within an expression operators of equal precedence are processed from left to right Examples Values in three PC wells 3 002 3 186 2 998 Values in three NC wells 0 868 0 798 0 824 PC NC 100 0 02232
5. e Reading a Plate With a Protocol e Reading Plate Data from a File e Simulating a Plate Read e Collecting Data from an EL301 Microstrip Reader Getting Started e 3 9 3 10 o Getting Started Data Reduction and Analysis After data reduction is performed on microplate data results are displayed in the Raw Data format Depending on the protocol definition Blanked Data Pathlength Correction Transformation Results Concentrations Cutoffs Template and or Plate Formula Results may also be displayed i KCjunior Results Protocols Utilities Setup Curent Reader View Help Resuts ejas Protocol joas Raw Data Blanked Data Pathlength Correction Transformation Results Concentrations Labels Template Plate Formula Results Comments Warnings Results calculation is structured as follows Raw Data Blanked Data Pathlength Correction gt Transformation Results Plate Formula Results Concentrations Cutoftfs e The basis for calculations begins with the Raw Data e If blank subtraction is performed on the Raw Data Blanked Data becomes the basis for calculating Transformation Results Plate Formula Results Concentrations and Cutoffs e If Pathlength Correction is enabled to normalize absorbances to 1 cm MultiWavelength reads only the corrected absorbances become the basis for calculating Transformation Results Plate Form
6. e Click the Spectral Chart button to select up to 8 spectral curves to overlay in one graph Spectral Chart Spectral scan results are shown below in the Spectral Chart format Raw Data Template Comments Warnings r Scan View Options z Calculation Options C Raw Data Spectral Chart Spectral Chart G Blented Data C Peak ODAVavelenath Specta Chat e In this format each well displays a graph of the data points corresponding to the OD readings taken across a wavelength range e To enlarge the view of the curve in an individual well place the cursor over the desired well and click the left mouse button e If blank subtraction was performed the spectral scan curves are automatically based on the Blanked ODs e Click the Calculation Options button to adjust the wavelength range used for calculations for all scanned wells e Click the Spectral Chart button to select up to 8 spectral curves to overlay in one graph e To enlarge the view of the curve in an individual well place the cursor over the desired well and click the left mouse button The Spectral Scan Curve dialog will appear 7 10 e Interpreting Results User s Guide to KCjunior Spectral Scan Curve Dialog Plate SAMPLE Scan Wel SMPO1 B1 Help Print Copy Options V Display Legend 360 420 480 540 600 660 720 780 840 900 Wavelength nm Peak OD 1 386 at 320
7. in the first Cutoff field Enter STD0O4 in the second Cutoff field Enter Neg in the first Label field Eqv in the second and Pos in the third Click the Reports tab Select Reports The Reports dialog is used to select and customize reports for this protocol L 2 Enter any text in the Report Banner field Check the Absorbance Report box and the accompanying Both radio button Check the Matrix Report box In the Options list highlight Well IDs then click Add In the Options list highlight Calculated Concs then click Add Check the Curve Report box Click OK to close the protocol Select Protocol Save Protocol Read a Plate E i 3 18 e Getting Started Click the Read Plate button Enter a Results ID using up to 16 alphanumeric characters Click Read Plate Place a microplate on the reader Note For best results gt Wells A1 and A2 should be empty gt The contents of wells B1 and B2 should result in a low optical density value at the defined Primary Wavelength gt C1 and C2 should read slightly higher and so on through H1 and H2 which should provide the highest OD Click OK User s Guide to KCjunior Interpret Results Following a successful plate read Raw Data Blanked Data Transformation Results Concentrations Labels and Template information will be displayed 1 Click on each of the six tabs to see results 2 Under Concentrations click Standard
8. 1 Enter a unique Name for the cartridge using up to 32 alphanumeric characters 2 Select the Cartridge Type Excitation or Emission For each filter wheel position enter the Wavelength and Bandwidth information or select Plug or Open gt Itis important that the wavelength and bandwidth information be entered correctly for each filter The wavelength and bandwidth values are often printed on the side of each filter gt Dispense Read protocols have special filter position requirements turn to the next page for more information 4 Click OK to save the changes and return to the previous screen or click Cancel to discard the changes User s Guide to KCjunior Setting Up the System e 4 13 Special Filter Position Requirements for Dispense Read Protocols Single filter set protocols e f light shuttering is specified a blocking filter Plug must be placed in the excitation filter wheel in one of the two positions next to the excitation filter that 1s specified in the protocol Dual filter set protocols e The two excitation filters specified in the protocol must be positioned next to each other in the excitation filter wheel e The two emission filters specified in the protocol must be positioned next to each other in the emission filter wheel e f light shuttering is specified two blocking filters Plugs must be placed in the excitation filter wheel 4 14 o Setting Up the System User s Guide to KCjuni
9. D gt If Graph Interpolation Result is enabled under Setup Configuration Reporting Options the interpolation value will be plotted on the curve The value will not be reported however if more than one sample is selected gt Data points cannot be edited within this dialog To exclude a well from the curve return to the Raw Data results display and Mask the desired well User s Guide to KCjunior User s Guide to KCjunior Click Print Copy to print the graph or copy it to the Clipboard Note A Titer curve report can also be printed if it is selected as a reporting option within the protocol gt Click Titer Results to view Curve Parameters R Square values and Interpolation results for all samples on the plate gt Toggle the Display Legend setting to include exclude a legend in the graph gt Click the right mouse button inside the graph to access advanced graphing options gt Click OK to close the Titer Curve dialog Titer Results Regardless of the number of titer curves selected for viewing the Titer Results dialog shows results for all samples defined for the plate Titer Results Dialog Curve Type Interpolation alue 4 Parameter y A D 7 1 x C B D aaa 1 000 Absorbance Help Sample ID Interpolation _ amp A 0 0167 B 1 7624 C 43 9181 D 5 0860 19 946 A4 0 0997 B 2 1114 C 108 4276 D 107 7871 11 293 Curve Fit Failed A4 0 2757 B 1 9834 C 101 4037 D 78 3194 9 625 A
10. MEAN PC NC 100 0 01946 User s Guide to KCjunior Defining Protocols e 5 53 Identifying Samples KCjunior provides the ability to assign custom identifiers such as Jones or 003672865A to sample groups These identifiers are referred to as Sample IDs and they are defined within the Sample ID Template To define Sample IDs 1 Ensure that Sample wells are defined appropriately within the Well ID Template 2 Access the Sample ID Template Protocol Definition General Information Read Method Template Curve Cutoffs Reports C Well lDs 0001 Dilutions 0001 SMPO3 SMPO Transformations SMP09 Sample IDs SMP17 0002 0002 SMP10 SMP10 SMP10 SMP18 0003 0003 SMP11 SMP11 SMP11 SMP19 0004 0005 0004 0005 SMP12 SMP13 SMP12 SMP13 SMP12 SMP13 SMP20 SMP21 IF SMPo6 SMPO6 SMPO6 SMP14 SMP14 SMP14 SMP22 G SMPo7 SMPO SMPO7 SMP15 SMP15 SMP15 SMP23 SMPos SMPO8 SMPO SMP16 Sample ID 0o06 ae SMP16 SMP16 SMP24 3 In this template highlight a sample well to prepare it for identification The well ID will appear in the Sample ID field Only one replicate within a sample group needs to be highlighted 4 Replace the contents of the Sample ID field with the new sample group identifier using up to 16 alphanumeric characters
11. Modifications to Software within a version Updates are expressly excluded from the terms of this Warranty If the Software is found to be defective by Bio Tek Bio Tek s sole obligation under this Warranty is to use efforts consistent with Bio Tek s regular business practices to attempt to remedy such defect In no event shall Bio Tek s liability under this warranty exceed the original purchase price of the Software This is a limited warranty This warranty contains the entire obligation of Bio Tek No other warranties express implied or statutory are given The implied warranties of merchantability and of fitness for any particular purpose are expressly excluded and do not apply to the software Under no circumstances shall Bio Tek be liable to the purchaser or any other person for any direct incidental or consequential damages whether arising out of breach of warranty or otherwise This warranty shall not be changed or modified in any way without the express written permission of an officer of Bio Tek Instruments Inc This Warranty shall be NULL AND VOID if any modifications are made to the Software by the Purchaser Correction of difficulties or defects traceable to Purchaser s errors or system changes shall be billed to Purchaser at Bio Tek s standard time and materials charge Licensing Agreement Document Revision LICENSE Single Use You have the non exclusive right to use the enclosed programs These
12. gt Click the eal button to the right of the Transformation Formula field to access the Formula dialog Formula Dialog Formula lt BLK1 A CTL1 BLK1 100 Functions Well IDs E a 3 Ed Verify Ea E st iS JE EEA EE E o es gt Locations cde ol ae gt Assemble the formula using a combination of Functions Well IDs Mathematical values and symbols and Location indicators A Function performs a specific operation on the contents inside the parenthesis such as the calculation of a mean value or the Standard Deviation The Well IDs list contains those Well IDs that are valid for this protocol Each Well ID represents the mean value of its group CTLI represents the mean of all wells labeled as CTLI The identifier X represents the current value of the well Defining Protocols e 5 47 gt Functions and Well IDs can be entered manually or they can be selected from the list boxes To add an item from a list box to the Formula field highlight the desired item then double click To insert an item between two existing items in the Formula field begin by clicking the mouse once in the Formula field where the new item should appear Highlight the desired item in the list box then double click to insert gt Mathematical values and symbols can be entered manually or selected by clicking the appropriate buttons gt Location indicators can be entered manually or selected by clicking the a
13. junior User s Guide User s Guide to KCjunior October 2002 Copyright 2002 PN 5271000 Revision E Bio Tek Instruments Inc Notices All Rights Reserved Trademarks BIO TEK INSTRUMENTS INC Highland Park Box 998 Winooski Vermont 05404 0998 USA Customer Service amp Sales Phone 888 451 5171 toll free in the U S 802 655 4040 outside the U S Fax 802 655 7941 E Mail sales biotek com Internet http www biotek com Service Technical Assistance Center TAC Phone 800 242 4685 toll free in the U S 802 655 4740 outside the U S Fax 802 655 3399 E Mail labtac biotek com European Coordination Center Bio Tek Instruments GmbH Kocherwaldstr 34 D 74177 Bad Friedrichshall Germany Phone 49 0 7136 9680 Fax 49 0 7136 968 111 E Mail info biotek de Copyright 2002 Bio Tek Instruments Incorporated This publication is protected by copyright and all rights are reserved No part of this manual may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying and recording for any purpose other than the purchaser s personal use without written permission of Bio Tek Instruments Inc Printed in the United States of America Bio Tek Part Number 5271000 Bio Tek is a registered trademark and KCyunior KCJr and OMNI are trademarks of Bio Tek Instruments Inc All other tradem
14. 5 Click the Apply button to assign the ID to the highlighted sample group 6 Repeat for the remaining sample wells 5 54 e Defining Protocols User s Guide to KCjunior User s Guide to KCjunior Curves KCyjunior provides three methods for generating curves A standard curve can be generated based on the standard wells defined within the protocol A previously generated and stored curve can be referenced from within the protocol A titer curve can be generated using multiple dilutions of sample groups as concentrations The basis for curve calculation may be Raw Data Blanked Data data normalized by Pathlength Correction or Maximum Slope depending on the protocol definition See Essential Information in Getting Started to review the results calculation structure To define Curve parameters L If this is a titer protocol ensure that samples and dilutions have been defined If the curve will be based on standards ensure that the Standard well IDs are assigned within the Well ID Template Note Each curve fitting method requires a minimum number of standards Linear 2 2 Parameter 4 Quadratic 3 4 Parameter 4 Cubic 4 Point to Point 2 Cubic Spline 3 Access the Curve dialog General Information Read Method Template Curve Cutoffs Reports Curve Method 4Parmeter ts Graph Report Name Sample Report M Y Axis Scale Linear Log Label Optical Density mX Axis Scale Linear C Log
15. Database directory within the KCjunior directory that you specified in step 13 Click Next to accept the default location or click Browse to specify a different location Note This location can be changed later from within KCjunior If a protocol database named PROTOCOL MDB already exists in this location the database is renamed and an empty PROTOCOL MDB is installed Question El 2 The file Protocol mdb already exists in this directory Do pou wish to owerste it Selecting Yes will cause the existing database to be renamed WS Protocol mdb gt Click Yes to rename the existing protocol database and continue Click No to return to the Choose Protocol Database Location dialog Installing the Software e 2 9 Data for each plate is stored in a Results Record in the Results Database This database is typically stored on a computer s hard drive or on a network to which the computer is attached 2 10 o Installing the Software After the protocol database location has been selected another informational dialog appears Information G Install will now ask for the location where you would like to store the RESULTS database IF You are unsure where it should be use the default location 16 Click OK to acknowledge the information The Choose Results Database Location dialog appears Choose Results Database Location x Select a directory where you would like the Results database to reside
16. Extrapolated concentration 10 6 Grayed numbers indicate a suppressed standard 10 9 User s Guide to KCjunior Illegal log function attempted 10 9 Insufficient valid standards 10 9 No valid blanks in sector 10 9 OUT 10 10 Pathlengths could not be generated 10 10 Plate was aborted during read 10 10 Reader control error occurred during read 10 10 Reader error 10 2 Reader Status Failure 10 2 Reader Timeout Error 10 3 Serial Read Error 10 3 Serial Write Error 10 3 Stored curve does not exist 10 11 Strikethrough indicates masked well 10 11 Too many standards for curve type 10 11 Unable to generate curve 10 12 Unable to initialize curve standards 10 12 Well read error 10 7 Well was set to maximum valid value 10 5 Well was set to minimum valid value 10 6 Excel exporting data to 6 21 Excitation filter 5 24 5 25 Excitation filter wheel 4 11 5 23 Extrapolate 5 57 7 2 Exporting data Copying results data and pasting it in another application 6 24 Delimiters 6 18 Directly to Microsoft Excel 6 21 Including an empty row or blank line 6 18 Including text headers 6 18 Specifying the first Excel cell location 6 18 To a text file 6 18 To an existing Excel spreadsheet 6 18 F Fast read 6 7 Fastest kinetic interval 5 31 Filter position requirements for Dispense Read protocols 4 14 Filter set FS function 5 51 7 19 7 20 7 27 7 29 Filter sets Auto sensitivity 5 24 5 28 Defining 5 24 Optics position 5 25 Options 5 27 Referencing in a
17. MPO SMPO MP04 SMP05 SMP06 SMP07 SMPO8 MP09 SMPIO SMP11 SMP1 We ID ee nad aoo aad aood aooe ao aood oo ood too coepi APO MPO MPO MPOd MPO APOG APO POS BOS MPIO MIP MIP We Bir mi mr a ir a rr a a MPO MPO P04 MPU6 MPO POE MPO9 MP10 MP MP We D MPO PO MPO POA MMPOG MPO MPO8 POG MPTO D A i i i ar ar e B i i E APO APO MPO APO EOE MP EOR APOS MPI0 MIP MIE We D 11 18 99 4 09 20 PM Page of 1 Protocol Report for a fluorescence assay showing the Template information User s Guide to KCjunior Appendix A Sample Reports e 11 3 Sample Report RAW DATA REPORT Protocol ID Sample Protocol 2 Plate Read Time 4 23 99 9 03 33 AM Plate ID Sample Plate 2 Temperature 36 9 C Warning Messages An M in front of a value indicates a masked well OUT indicates a well exceeded the min max range 0 456 0 471 0 668 0 735 1 505 0 843 0 633 0 634 0 921 l 0 699 054 Raw Daia S oos oos 030s ozn rop 037 ors orm oas ies 0 234 1 500 Bikdpad x 7504 TH 46 86 618 KASS Raw Data gt ow ond osd oss oso oz oss is osad iis 022d 0754 picapaa AA AA TOG O70 T3 ESE Raw Dats oad oad ond 0509 0251 osd oms isol osd o ae pl Di 90 hE T018 AQ oF 80 044 OTF E E i ra ory osmosed e e E onl oar oan mda 08 BY 70 E 300 of iE OO Raw Data crs os oms osa osi oso osal osa ossa oso ost 0837 Bikdpaa T 7 07 RR 7 KA KZ vE TIK VEET Raw Datal o on ond owd o a E pk Dara 449 7 88 Raw Date 1 985 0 424 at io 1 932 a 0 669 ine 1357 I 3
18. Mis Settings 8 o 3 3 Shut Down User s Guide to KCjunior Installing the Software e 2 3 3 When the Control Panel folder opens double click on the Add Remove Programs icon File Edit View Help D Jbt DBC Accessibility Add New S t sueus Date Time Options Hardware Prograrns 4 When the Add Remove Programs Properties dialog opens highlight KCjunior then click Add Remove Add Remove Programs Properties Install Uninstall windows Setup Startup Disk sI To install a new program from a floppy disk or CD ROM drive click Install Install The uninstall process leaves Tee The following software can be automatically removed by your protocol and results Fr Windows To remove a program or to modify its installed databases in their original components select it from the list and click location s on your Add Remove computer Cadra for Windows Corel Uninstaller Doc To Help 4 0 Fax Sr Print to M ail 3 0 HTML Help Workshop KC4 2 6 Rev 3 McAfee VirusScan v4 0 3 Licensed Microsoft Internet Explorer 5 and Internet Tools Add Remove OF Cancel Apply 5 Follow the instructions on the screen to complete the uninstall process 2 4 e Installing the Software User s Guide to KCjunior Software Installation To l A see ee T User s Guide to KCjunior Install KCjunior Start Windows e Note If you are prompted with the Windows password entry enter a valid pass
19. Note For the EL311 312 340 readers select only one well Click OK to save the settings and return to the Read Method parameters dialog Defining Incubation Parameters If the currently selected reader supports plate incubation the Incubation option is available To define Incubation parameters L Ensure that under Setup Reader the desired Reader Type is currently selected gt The reader does not need to be connected to the computer to define Incubation parameters Check the Incubation box The Temperature field becomes accessible Enter the setpoint temperature C in the Temperature field This is the temperature to which the plate chamber will be heated prior to plate reading The valid range is from 20 C or 4 C above room temperature whichever is greater to 50 C Defining Shake Parameters If the currently selected reader supports plate shaking the Shake parameters are accessible in the Read Method dialog To define Shake parameters i Ensure that under Setup Reader the desired Reader Type is currently selected gt The reader does not need to be connected to the computer to define Shake parameters Select a Shake Method from the drop down menu Available options include None Shake First Read and Shake Every Read gt If Shake First Read is selected the plate is shaken prior to the first plate reading gt If Shake Every Read is selected the plate is shaken prior to every plate rea
20. Plate Read Time field gt The possible Plate Status values are OK if the plate was read without error Aborted if the plate read was aborted before completion Reader Control Error if the reader experienced a failure before the completion of the read and could no longer respond to KCjunior Reader Error code if the reader experienced a failure before the completion of the read and was able to communicate an error code gt The Reader field contains the reader model with which the plate was read gt The name of the person to whom KCjunior is registered appears as the default in the Created By field The name can be changed here if the plate has not yet been saved gt If applicable the Temperature will be reported in degrees Celsius This value reflects the temperature of the incubation chamber at the time the plate was read gt If the Monitor Well feature was enabled in the protocol the dialog displays the elapsed time between the start of the User s Guide to KCjunior Interpreting Results e 7 35 7 36 e Interpreting Results gt gt monitoring process and the point when the monitored well s met the specified criteria The Plate Description can be defined or edited in this dialog To see the Read Method parameters defined for this plate click the Read Method button Click OK to save any changes and return to the previous dialog Click Cancel to close this dialog without saving changes Well In
21. Protocols for more information User s Guide to KCjunior Appendix A Sample Reports e 11 1 Sample Report PROTOCOL TEMPLATE REPORT Plate Read Time 4 23 99 8 47 27 AM Temperature 37 0 C Protocol ID Sample Protocol 1 Plate ID Sample Plate 1 General Information Protocol Description This report contains sample data You can enter a Protocol Description using up to 1024 alphanumeric characters Raw Data Limits 3 000 to 3 000 gt I Discard Out of Range Values Blank Data Limits 3 000 to 3 000 X Discard Out of Range Values Decimal Points 3 Scientific Notation Raw Data Blanks Scientific Notation Transforms Concentrations Blank Method Full Read Method Information Chemistry Absorbance Read Method Endpoint Primary Wavelength 405 nm Reference Wavelength Geometry Delay Before Read Incubation Shaking Curve Information Curve Method Graph Name Y Axis X Axis Average Standards n a 8x12 10 secs 37C Before first read low intensity 30 secs 4 Parameter Cytotoxicity Linear Absorbance Linear mmol Yes Extrapolate Yes Interpolation Value 0 100 Transformations Transformation Formula A 2 A 12 2 A 12 E AE ANE E 3 e 589 NAMIC E 2 E 12 2 E 12 e F 2 F 12 2 F 12 G 2 G 12 2 G 12 H 2 H 12 2 H 12 Plate Formula xX 100 Cutoff Information Cutoff STDO2 STD04 STD06 STDO8 11 18 99 4 04 36 PM Page of 1 Pr
22. Read 2 Click the Dispense Read button 3 Ifaread will be performed check Perform Read If no read will be performed skip to step 8 4 If performing a read choose Well Mode or Plate Mode gt In Well Mode the FLx800 reads each well individually for a specified number of times using 1 or 2 sampling windows dispensing reagent as specified in Dispense Parameters see step 8 This mode is typically used for fast kinetic reactions gt In Plate Mode the FLx800 reads a whole or partial plate for a specified number of times dispensing reagent as specified in Dispense Parameters see step 8 Defining Protocols e 5 21 10 11 12 5 22 e Defining Protocols If reading in Well Mode select the number of Sampling Windows or 2 For one or both Sampling Windows gt Enter a Lag Time from 0 to 600 seconds This is the delay before the first Sample is read Note For two Sampling windows KCjunior automatically calculates the Sampling Start time for the second window The second window begins immediately after the first finishes unless a Lag Time greater than 0 is specified for the second window gt Enter the total number of Samples measurements to be taken per well from 1 to 300 gt Enter the Sample Interval the delay between sample measurements from 0 02 to 12 seconds gt Optional Check Light Shutter to block the light when not measuring to inhibit photo bleaching If reading in Plate Mode
23. Release the mouse button before releasing the Ctrl button To zoom in on a portion of the graph and have the axes adjust accordingly 1 Press and hold the Shift key 2 Press and hold the left mouse button 3 Drag the mouse to select the zoom area 4 Release the mouse button before releasing the Shift button Advanced Graphing Options Clicking the right mouse button from within the graph provides access to many advanced graphing options including e Creating a border around the entire chart e Changing the color of the background foreground lines and symbols e Scaling the X and or Y axis to better focus in on the curve User s Guide to KCjunior e Changing the title and font of the X and or Y axis e Adding a legend to the graph e Adding informative labels to the graph Printing and Copying the Graph The graph and any edits made to it can be copied to the clipboard or printed To print or copy the graph 1 Click the Print Copy button A selection list will appear poms Ca po Copy Chart to Clipboard Cancel 2 Click the Print Chart button to print the graph Click the Copy Chart to Clipboard button to copy the graph to the Clipboard The graph can then be pasted into another program 4 Click Cancel to return to the graph Editing the Standard Curve After the standard curve has been generated one or more standards can be temporarily excluded from the recalculation of the curve This
24. To install in this directory click Next To install in a different directory click Browse and select another directory Click Cancel to exit the Setup program 17 The default Results Database Location is the Database directory within the KCjunior directory that you specified in step 13 Click Next to accept the default location or click Browse to specify a different location Note This location can be changed later from within KCjunior If a results database named RESULTS MDB already exists in this location the database is renamed and an empty RESULTS MDB is installed User s Guide to KCjunior 2 The file Results mdb already exists in this directory Do you wish bo overvrite it e After the results database location is selected the software installation begins 18 Toward the end of the installation the Select Groups dialog appears Select Groups Setup will add items to the group in the Program Group box YOU can Wipe a new group name or select one From the existing Groupe list Program Folders Chun Existing Folders Accessories Adobe Acrobat 4 0 Cadra for Windows CorlDR aw amp Fas Sr Print to h ail HTML Help Worksho MoAtee VirusScan Back Cancel 19 Click Next to accept the default program folder name or type in a new one 20 When Setup is complete KCjunior instructs you to restart your computer e KCyjunior will automatically restart your computer reco
25. User s Guide to KCjunior Setting Up the System e 4 1 e To switch between the two readers use the Current Reader Menu KCjunior Results Protocols Utilities Setup gE Select Filter Wheel Cartridges Get Curent Reader Status gt KCjunior automatically switches readers if a protocol is opened that is not compatible with the currently selected reader Connecting the Reader to the Computer Before KCjunior can communicate with a microplate reader the reader s must be properly connected to the computer on which KCjunior is installed The following steps briefly describe how to connect the reader to the computer For more detailed information refer to the reader s user manual 1 Attach the appropriate cable to an available serial port on the back of the computer gt If you are going to configure KCjunior for use with two readers the computer must have two serial ports 2 Plug the other end of the cable to the serial port on the reader Connect the power cable to the back or side of the reader Plug all power cords into grounded electrical outlets gt Use of a surge protector is strongly recommended 5 Use KCjunior to set up the communication parameters as described in the next section Reader Setup 4 2 e Setting Up the System User s Guide to KCjunior Reader Setup Important Set and test communication parameters before attempting to read a microplate Follow these steps in order L For communicat
26. gt Enter the total number of Kinetic Reads from 1 to 300 gt Enter a Kinetic Interval delay between reads of 1 to 9999 seconds gt Optional Check Light Shutter to block the light when not measuring to inhibit photo bleaching If performing a dispense check Perform Dispense Optional If no read will be performed or if a Well Mode read will be performed choose a Tip Prime Option of Before First Well or Before Every Well and then enter a dispense tube Prime Volume of 5 to 20 ul gt Tip If there is a long duration between dispenses from well to well consider priming Before Every Well to compensate for any fluid loss at the dispense tip due to evaporation Optional If a Plate Mode read will be performed check Prime Tip Before Start of Processing and then enter a dispense tube Prime Volume from 5 to 20 ul Enter a reagent Dispense Volume from 5 to 1000 ul gt The minimum Dispense Volume varies depending on the Dispense Rate selected Select a fluid Dispense Rate of 1 2 3 or 4 gt With each rate selection the corresponding ul sec value is displayed User s Guide to KCjunior User s Guide to KCjunior 13 14 15 Optional If no read will be performed or if a Well Mode read will be performed enter a Lag Time from 0 to 6000 seconds This is the delay before the dispense gt Tip If two Sampling Windows are defined and you wish to dispense between the two windows enter a Lag Time that
27. if not previously assigned To close an open record select Results Close Results or click the toolbar icon If the record is new or if modifications were made to an existing record KCjunior provides an option to save AN Save new results e Click Yes to save the new or modified information and close the record e Click No to close the record without saving changes e Click Cancel to close this dialog and return to the main view The new or modified information is retained but not yet saved Copying an Existing Results Record KCjunior provides an option to copy all of the contents of an existing results record to a new record with a different name To copy an existing record begin by opening it Once it is open immediately select Save As from the Results Menu KCjunior asks for a new name Results Save As Dialog Results ID E ligonucleotidel Cancel e Enter a new name then click OK to create the new results record save it in the current Results Database and keep it open for modification e Click Cancel to cancel this operation and return to the original results record User s Guide to KCjunior User s Guide to KCjunior Sharing Results Records and Databases Since KCjunior stores results records in a database there are several options for sharing records and databases among users A results database can be stored on a network for multi user availability gt Each user must configure the Resu
28. matches the calculated Sampling Start time If a Plate Mode read will be performed enter an Inject on Interval value from 1 to the number of Kinetic Reads This is the read number at which the dispense will occur gt Note The kinetic curve will display a green line on the X axis Time to indicate when the dispense occurred Click OK to save the changes and return to the Read Method dialog gt Important If tip priming will be performed make sure that the tip priming trough is installed in the instrument refer to the operator s manual for placement instructions Selecting Filter Wheel Cartridges For a FLx800 that is used for a variety of applications multiple Excitation and or Emission filter wheel cartridges may be prepared and installed as needed The contents of Excitation and Emission filter wheel cartridges are defined under SetuplConfiguration You can then select the cartridges to be associated with each protocol To select filter wheel cartridges 1 Ensure that under Setup Configuration Filter Wheel Cartridges Excitation and Emission cartridges are defined Click the Select Cartridges button from the Read Method dialog The Select Filter Wheel Cartridge dialog appears Select Filter Wheel Cartridge Dialog x Excitation Filter Wheel Ex Cartridge 1 View Cancel Emission Filter Wheel Help View Using the drop down menus select the desired Excitation and Emission Filter Wheel cart
29. see Reader Support in Setting Up the System for more information Selection criteria includes fluorescence absorbance reading capabilities and read method parameters If the readers are equally capable of supporting the protocol KCjunior does not change the Current Reader e To close this dialog without opening a protocol click Cancel or press the Esc key e To select a different Protocol Database close this dialog then select Setup Configuration Pathnames Creating a New Protocol To create a new protocol begin by clicking the New Protocol button in the KCjunior main screen selecting New Protocol from the Protocol Menu or by clicking the toolbar icon The General Information dialog appears requesting identification information for the new protocol See Defining General Information at the start of Defining Protocol Parameters for more information Before Defining a New Protocol The available definable parameters for every new protocol vary depending on the currently configured Reader Type Note Before defining a new protocol make sure the Reader Type is properly set under Setup Reader and the correct reader is selected under Current Reader Examples e If the currently selected Reader Type is ELx808 the available Read Method Types are Endpoint and Kinetic e If the currently selected Reader Type is PowerWave 340 the available Read Method Types are Endpoint Kinetic and MultiWavelength and the avail
30. than the PowerWave XS Do not select PowerWave340 or PowerWave340I for a PowerWavexX 340 instrument gt KCyjunior can be configured to communicate with any two Bio Tek readers if the computer has two serial ports See Reader Support in Setting Up the System for more information Click OK to continue If necessary KCjunior will provide instructions for setting additional communication parameters See Reader Setup in Setting Up the System for more information User s Guide to KCjunior Where to go from here e See Password Distribution and Software Registration on page 2 16 to find out how to ensure continuous use of KCjunior after the 30 day trial period and how to register your software with Bio Tek Instruments e Review the next section Getting Started to gt gt gt gt Learn about KCjunior s menu structure and how to navigate around the Help system Review essential information what you should know before using KCyunior Get started with the Quick Start Take a guided tour to learn how to create protocols read plates interpret results and more e Examine sample protocols and results records and read the corresponding assay descriptions gt gt User s Guide to KCjunior From the KCjunior main screen select Help Help Topics Double click on Sample Protocols and Results Records then double click again on KCjunior Samples The KCjunior Samples help will a
31. 0 enter a wavelength from 1 to 999 or leave it set to 0 if no reference wavelength is required gt Ifthe Kinetic Interval field is accessible enter a value in seconds from 1 to 9999 gt Click OK to accept the values KCjunior validates the settings and then calculates the results 7 To save the results select Results Save Results or press CTRL S 8 KCjunior requests a Results ID if one was not defined before the plate read Enter the ID using up to 32 alphanumeric characters This is the name under which results will be stored in the Results Database 9 Select OK to save the results Collecting Data from an EL301 Microstrip Reader The EL301 microstrip reader does not support computer control for its operation Optical density data stored in the reader however can be transferred to KCjunior for analysis e KCyjunior accepts EL301 data in Absorbance ABSORB mode and does not accept data in the Ratio RATIO Linear Equation LINEQU or Difference DIFF modes e KCyjunior accepts data for up to 96 wells with each transfer gt To transfer two or three plates of data to KCjunior send them separately and save each plate under a different name e Ifa blank well was read the EL301 automatically performs blank subtraction before data transfer e Ifa dual wavelength read is performed the EL301 automatically calculates the Delta OD before data transfer To collect data from an EL301 reader 1 Perform the n
32. 0 244 0 182 Statistics Standard Curve Powerwavex Com2 Sim The Raw Data set of results is always displayed Raw Data represents the measurements that were either retrieved from the microplate reader entered manually or read from a file Results are arranged with rows and columns that align with the specified plate geometry Depending on the protocol definition Blanked Data Pathlength Correction Transformation Results Concentrations Labels Template and Plate Formula Results may also be displayed Interpreting Results begins by describing the Well Notations that may appear in one or more results categories then explains how to interpret each of the eight possible sets of results Interpreting Results e 7 1 Well Notations 7 2 e Interpreting Results Within the results displays different circumstances may cause the result in a well to be displayed with a special symbol such as a strHke through or parenthesis or to be completely replaced with symbols such as The following list describes the well notations that may appear in results displays and on reports If a well contains OUT in place of an OD RFU or RLU the raw data value fell outside the acceptable min max data range defined in the protocol and it has been excluded from further calculations gt See General Information in Defining Protocols for information on setting Raw Data Limits and Blank Limits If a
33. 2 5 Tons CO esa E a aia ate dea noe saul ieedends 2 5 TOS CARE IC UMON aie create ras Peicelecarcede ond a aa 2 12 W Here 10700 ArOmm NETO mos raana catia lau a aA aeeda r udebanateeaeeien Ts 2 15 Password Distribution and Software Registration cccccccesseesessseeeeeceeeeeeeeeaeseeeeseseeseeeeess 2 16 Obtaining a password and registering the software online cccceeeeeeeeeeeeees 2 17 Obtaining a password if you do not have Internet acceSs ccscsceeeeeeeeeeeeeeeees 2 17 Registering the software if you do not have Internet access cceeeeeeeeeeeees 2 17 Getting Started 3 1 KeCjumior s Ment SOc cs 255 sias leseenictee cos che E O E E ARERR 3 1 TPOOIDA enean a a a a E 3 2 ReSWlie ME rere E A E 3 3 POO COLMO a A E E A E 3 4 UES MEN e a a a a a 3 5 SOUP Monina ar n A r OSS 3 6 Current Redder Menu soera neen a a a a 3 6 E A CUM E E E cate E E E E A E E E A E A A A E 3 7 N e a E E raennanuete ican 3 7 Essential ITniorman OW essas ninnaa nnna n a A A A E A Oar aeS 3 9 Protocol DeHnitiOm renee e a R 3 9 Plate Datar A cguis OMaria ane aa a E ETE 3 9 Data Reduction and Analysis ccccsssssssssseecccccccecccceaceeeesssseeeseceeceeeeeseaaaaegeessses 3 10 OU ESAE a a a A 3 11 Guided TOUT erras E E EEE E E E NEOR 3 12 User s Guide to KCjunior Contents e i Setting Up the System 4 1 Reader SUD POL eeni tcarathaetaasies ie ads aad saas verdana nian e dawn iausacanaeaadieet anor A 4 Wal Reader SUP POR
34. 36 Index e 13 5 Kinetic Interval 5 31 Kinetic Reads 5 31 Monitor well 5 32 Number of Plates 5 10 5 20 Plate Geometry 5 31 Read Method Type 3 11 5 10 Sampling 5 27 Sensitivity 5 26 Total kinetic read time 5 31 Validating parameters 5 31 Wavelengths 5 16 Read mode selecting 5 35 Reader 1 or 2 4 1 Reader calibration 5 37 6 8 Reader errors 7 35 10 2 10 10 Reader setup Baud Rate 4 4 Com Port 4 4 Data Bits 4 4 EOT Character 4 4 Get Comm Defaults 4 4 Parity 4 4 Reader Type 2 14 4 3 5 10 Stop Bits 4 4 Test Communications 4 5 Reader system test 9 8 Reader system test history 9 9 Reader type 2 14 4 3 5 10 Reading a plate 6 13 6 15 Reading a plate with a protocol 6 10 Reading a plate without a protocol 6 7 Reading plate data from a file 6 3 6 13 Reconfiguring pathnames 4 5 4 10 5 2 5 6 Reference wavelength 5 16 Referencing a well location in a formula 5 52 Registering KCyunior 2 15 Relative fluorescence luminescence units 12 1 Removing KCjunior from your computer 2 3 Report options 5 8 Reports Available types 5 61 11 1 Display curve legend 4 9 Graph interpolation result 4 9 Print preview setup 8 1 8 3 Print results 8 2 Printing 8 1 Sample printouts 11 1 Selecting in a protocol 5 61 Results Analyzing 6 24 Auto sensitivity results 7 32 Blanked Data 7 16 Concentrations 7 20 13 6 e Index Cutoff values 7 27 Database 4 9 4 10 6 2 Labels 7 27 Manipulating 7 34 Pathlength correction 7 17 Plate Formula Resu
35. 5 Simulating a plate read 6 3 6 12 Simulation data 6 3 6 12 6 24 7 34 7 38 Software registration 2 15 Software serial version number 1 3 Spectral scan Adjusting the wavelength range used for calculations 7 12 Calculation options 7 10 Calibrate before first read 5 37 Curve display options 7 12 Peak OD 7 11 Protocol definition 5 20 Raw and blanked spectral data 7 9 Spectral chart 7 10 Standard curve Editing 7 25 Printing 5 61 7 25 Storing 7 26 Titer curve 7 22 Viewing 7 21 Standard deviation SD 5 52 12 1 Standard outliers editing 7 21 7 25 Standards defining 4 6 5 38 Starting protocol 4 9 Statistics 7 30 Status Plate status values 7 35 Read status values 7 37 Stop bits 4 4 Stopping a read 6 9 6 11 10 9 Stored curves Archiving 9 8 Deleting from database 9 21 Use stored curve 5 56 Storing a standard curve 7 26 Subtraction in a formula 5 50 User s Guide to KCjunior Surge protector 4 2 Sweep mode 5 35 Symbols in results 10 5 System configuration 4 5 5 39 System requirements minimum 2 2 System test reader 9 8 T Technical support 1 3 Temperature setting 5 34 9 1 9 2 Template Assigning well Ds 5 38 Dilutions 5 44 Sample Ids 5 54 Transformations 5 46 Viewing results 7 28 Test well range 5 29 Testing communication 4 5 Tip prime option 5 22 Titer Curves 7 22 7 26 Defining dilutions 5 44 Interpolation value 5 57 Statistics 7 30 Top optical probe 5 25 Total kinetic read time 5 31 Tr
36. Bits value to 2 Specify an EOT Character This represents the character sent by the reader to KCjunior to indicate the end of transmission of a plate of data gt The default setting is 26 001A Z gt For the EL311 EL312 and EL340 readers the EOT Character may be changed The setting in KCjunior must match that on the reader gt For the EL301 microstrip reader the EOT Character has no significance gt The FLx800 does not require an EOT Character gt For all other readers the EOT Character must be set to 26 When all parameters are set click the Test Communications button to attempt to establish communication with the reader gt If the test is successful the communication parameters are set correctly gt If the test is unsuccessful appropriate instructions are provided for fixing the communication parameters Click OK to save the settings close the Reader Setup dialog and return to the KCjunior main screen User s Guide to KCjunior System Configuration Various system default parameters can be configured within the Setup Configuration group of dialogs Configurable parameters include well identifiers several reporting options results and protocol database pathnames and well type colors Well Identifiers KCjunior supports four different types of wells Blank Standard Sample and Control and processes each type as follows e The average of the Blank wells is automatically subtracted f
37. Condition 2 Success If Condition 1 is not met after a sensitivity reading of 250 or before the sensitivity at which one or more of the High Wells exceeds the High Value then the sensitivity of the highest valid signal to noise ratio acheived is chosen for the actual filter set read If the highest signal to noise ratio occurs at multiple sensitivity readings then the highest sensitivity is selected Condition 3 failure If Condition 2 is not met because there are no valid signal to noise ratios then an error message will appear and the read will be terminated Interpreting Results e 7 33 Condition 4 failure If none of the previous conditions are met then an error message will appear and the read will be terminated Signal to Noise Ratio Calculation o Ifthere are multiple Low Wells and Test Wells defined in the protocol the signal to noise ratio is calculated as follows Mean of the Test Wells Mean of the Low Wells Total Standard Deviation where Total Standard Deviation SQRT Std Dev of the Test Wells 2 Std Dev of the Low Wells 2 e If there is a single Low Well and or a single Test Well the signal to noise ratio is calculated as follows Mean of the Test Wells Mean of the Low Wells Mean of the Low Wells Results Options From within any results matrix highlighting a well and clicking the right mouse button produces a sub menu with a variety of options Plate Information 0 000 Well Infor
38. Control wells are defined appropriately within the Well ID Template 2 Access the Dilutions Template Protocol Definition General Information Read Method Template Curve Cutoffs Reports C WelllDs SMP02 Dilutions SMPO03 SMPO04 SMPOS Transformations SMPO C Sample IDs SMP 10 SMPO2 SMPO3 SMPO4 SMPOS5 SMPO SMP10 SMPOS ir SMPOS aes SMPOS SMPOS je la u SMPO6 s 18 SMPO SMP10 i eanan SMPF10 4S BES SMP10 16 116 SMP02 32 16 SMPO03 32 116 SMP04 64 m SMPo1 sMPo2 SMPO3 SMPO SMPOS Start Dilution 2 SMPO1 SMPO2 SMPO3 SMPO4 64 64 64 6 h a C Down m Replicates Direction Across Fill Count Delta IV Factor Down Map Direction C Actoss User s Guide to KCjunior 3 To assign dilutions and manually increase the dilution values gt gt gt Enter the starting dilution value in the Dilution field The valid dilution range is from 1 0000e 99 to 9 9999e 99 Specify the Number of Replicates and Replicate Direction The Number of Replicates represents the number of contiguous wells to be assigned the same dilution value The Replicate Direction is the direction in which contiguous wells should be assigned the same dilution value Down or Across Within the template click one or more wells to assign the cur
39. Curve to see the standard curve 3 Under any tab click Statistics to see statistical information for every Standard Sample and Blank group Print Reports and Store Results 1 Ifa printer is connected to the computer either directly or via a network select Results Print Results to print reports 2 To save the results record select Results Save Results The record will be stored under the Results ID in the Results Database User s Guide to KCjunior Getting Started e 3 19 3 20 e Getting Started User s Guide to KCjunior Setting Up the System Reader Support KCjunior supports most of Bio Tek s automated microplate readers including EL301 ELx800 PowerWave 200 EL311 EL808 PowerWave 340 EL312 ELx808 PowerWaveX EL340 FLx800 PowerWavexX 340 EL800 UQuant PowerWavexX Select PowerWave XS Dual Reader Support KCjunior can be configured to communicate with any two Bio Tek readers if the computer has two serial ports This allows you to for example connect an absorbance reader and a fluorescence reader to one computer and switch easily between them from within KCjunior Note If you have a laptop computer you may need to install a communications card e To configure KCjunior s communication parameters start by selecting Reader 1 or Reader 2 from the Setup Menu as shown below a KCjunior Plate Protocol Utilities FRO Configuration gt See Reader Setup on page 4 3 for further instructions
40. For multiple Filter Set reads curves are based on the measurements at the first defined filter set unless Transformation Formulas utilizing the FS function are applied to specify a different filter set e See Defining Transformations Formula Syntax in Defining Protocols for more information on the WV and FS functions e Click the Standard Curve button to view the graph see next page 7 20 e Interpreting Results User s Guide to KCjunior To access advanced graphing options place the cursor inside the graph and click the right mouse button User s Guide to KCjunior The Standard Curve The Standard Curve dialog displays the calculated curve and provides options for printing copying and storing the curve and for editing standard outliers and or curve parameters Standard Curve Dialog o Q C G 2 o a 2 SAMPLE Standard Curve Help Print Copy Edit Stds s Standards Ses angel tore th Ss gt Suppressed Stds Store the Curve Standard Curve M Display Legend 30 Concentration Linear y Ax B 4 0 0453 B 0 1177 R Square 0 9688 Click Print Copy to print the graph or copy it to the Clipboard gt A Curve Report can be printed if it is selected as a reporting option within the protocol See page 5 61 for more information If the Calculate Interpolation Value curve parameter is selected in the protocol click Interpolation to see the concentration calculated fo
41. Get current reader status 3 6 Get reader wavelengths 5 16 5 36 Index e 13 3 Getting Help 3 8 Glossary 12 1 Graph Copying 7 23 7 25 Moving scaling zooming 7 24 Printing 5 61 7 23 7 25 Graph interpolation result 4 8 Graphing options Axes scaling 7 24 Borders and colors 7 24 Legends and labels 7 24 Spectral scan curve display options 7 12 Guided tour 2 15 3 12 H Help menu 3 7 Help system 3 8 High value 5 30 High well range 5 29 I Importing Protocols 5 6 Results records 6 5 6 6 Incubation Defining read method parameters 5 34 Incubation control utility 9 2 Override button 9 3 Setting a delay before reading 5 32 Inject on interval 5 23 Interpolation Calculate interpolation value 5 57 Graph interpolation result 4 9 Interpolation result 7 21 7 22 7 23 K KC Jr for DOS plate files opening 6 3 6 13 Kinetic Fastest interval 5 31 Interval 5 31 Max slope unit of measure 7 8 7 9 Reads number of 5 31 Kinetic read 5 10 Defined 12 1 Results 7 5 13 4 e Index L Labels 5 58 7 27 Lag time 5 22 Legend displaying in graph 4 9 7 21 7 22 7 23 Lin or Log axes 5 56 5 57 7 24 7 25 Linear curve 5 56 Logit Log curve 5 56 Low well range 5 29 Luminescence Auto sensitivity results 7 32 Blanked Data 7 16 Chemistry 5 21 Dispense Read 5 10 5 21 Endpoint read 5 10 Kinetic read 5 10 Raw Data 7 4 Relative Luminescence Unit RLU 12 1 M Masking reads wells 7 37 Maximum slope Curves 3 11 5 5
42. Label Concentration Average Standards Extrapolate Calculate Interpolation Value Use Stored Curve Cancel Defining Protocols e 5 55 5 56 e Defining Protocols If the standard curve will be based on a stored curve gt Click Use Stored Curve at the bottom of the Curve dialog gt Select a stored curve from the drop down list gt No other Curve parameters need to be defined Select a Curve Method from the drop down list Available options are Linear Quadratic Cubic 2 Parameter 4 Parameter Point to Point and Cubic Spline Linear A simple best fit straight line is plotted using the values of the standards Quadratic Uses the quadratic equation ax bx c y to plot the standard values Using this curve any particular data point for a standard which deviates from the ideal value does not affect the entire curve Cubic Uses the equation ax bx cx d y to plot the standard values The cubic curve is affected even less than the quadratic fit when any particular standard has a poor value 2 Parameter Also known as Logit Log Characterized by a skewed sigmoidal plot that eventually becomes asymptotic to the upper and lower standard values The logistic equation is algebraically transformed to a simpler form where experimentally determined values are used for the responses at concentrations of zero and infinity 4 Parameter Characterized by a skewed sigmoidal plot that eventually becomes asympto
43. Read Method parameters then click OK to continue The Simulation Data dialog appears in a format corresponding with the defined Plate Geometry 7 Enter OD RFU or RLU values directly into the appropriate wells or copy values from the spreadsheet and paste them here by pressing Ctrl V gt Ifa dual wavelength Endpoint or Kinetic absorbance read is defined the value entered in each well must represent the delta OD for that well gt Set the Kinetic Read value to simulate each plate reading in the kinetic reading cycle gt Set the Wavelength value to simulate each wavelength reading in a MultiWavelength read 8 When the data entry is complete click OK KCjunior performs data reduction based on the defined protocol parameters if applicable and then displays the results User s Guide to KCjunior Head From File User s Guide to KCjunior Reading Plate Data from a File KCjunior can read plates that were generated with Bio Tek s KCur for DOS software and absorbance files generated by Bio Tek s OMNI System To read plate data from one of these files L If desired create a protocol or open an existing one The read method parameters should be appropriate for the file to be opened Note Before creating or defining a protocol to accommodate a multi wavelength Omni System absorbance file make sure the reader type specified under Setup Reader supports multi wavelength reads The reader does not
44. Select Plate Print Results to print reports Select Plate Save Results to save the information Select Utilities Database Maintenance to archive protocol and plate data Getting Started e 3 11 Guided Tour Welcome to the Guided Tour to KCjunior This tour takes approximately 20 minutes and shows you how to do the following Prepare the microplate reader Define and test communication parameters Create a simple quantitative protocol Design Well ID Dilution Transformation and Sample ID templates Define curve fitting parameters Define cutoff criteria Select reports Read a plate Interpret results Print reports and store results Let KCjunior s Help system Refer to the User s Guide when taking the tour or let the KCjunior Help guide you on the tour system guide you To access the guided tour in the help system 1 Start KCjunior 2 Select Help Help Topics from the main screen 3 Double click on Getting Started 4 Double click on Guided Tour 5 Double click on H Guided Tour then follow the directions If the Help window is in your way you can move and resize it as you take the tour It will stay on top of KCjunior Prepare the Reader Refer to the reader s user manual for assistance if necessary 1 Ze 3 12 o Getting Started Connect the reader to the computer and turn it on Through the reader s front panel find the Baud Rate Data Bits Stop Bits and Parity informati
45. Time g 00 03 45 Options Options Plate Geometry ext 2 Read Al H12 M Eject between filter setreads IV Monitor Well Monitor Well Parameters e During a Kinetic Read the plate is read a defined number of times using one or two filter sets One Relative Fluorescence Unit RFU or Relative Luminescence Unit RLU is reported for each well for each kinetic read and for each filter set A graph showing RFU RLU vs Time is generated for each well User s Guide to KCjunior Defining Protocols e 5 13 Protocol Definition Dispense Read l d Validate Read Method _ J Eject between filter set reads Jalase M il ja l Shake Memad MGHILGT WEN Petenietens ters Darana WORURUGUS SHAKE r e During a Dispense Read sequence FLx800 fluid is dispensed before or during sample measurement KCyjunior generates a kinetic curve showing Relative Fluorescence Units RFU Y axis versus Time X Axis for each well 5 14 e Defining Protocols User s Guide to KCjunior Defining Read Method Parameters After the Read Method Type is selected the definable parameters automatically adjust accordingly All possible Read Method parameters are described on the following pages See below for a list of the remaining topics in this section e The following topics are specific to absorbance readers Selecting Primary and Reference Wavelengths p 5 16 Selecting Multiple Wavelengths
46. User s Guide to KCjunior Glossary List of Terms User s Guide to KCjunior Abscissa The coordinate representing the distance of a point from the y Axis in a plane Cartesian coordinate system measured along a line parallel to the X AXIS Ambiguous Curve A curve is ambiguous if multiple concentrations exist for a given OD RFU or RLU value Average Standards In a protocol if standard groups are defined with multiple replicates the Average Standards option is available e If Average Standards is enabled KCjunior uses the average of the standard replicates when generating the curve e If Average Standards is disabled KCjunior uses the individual standard replicates when generating the curve Coefficient of Variation CV The coefficient of variation represents the standard deviation expressed as a percentage of the mean Standard Deviation Mean 100 In formulas the coefficient of variation is represented by CV Glossary e 12 1 Database A database is a computer file that contains multiple records of information In KCjunior e The protocol database contains multiple records called protocols Within each record protocol is a set of fields containing information specific to that protocol such as protocol description read method type and curve method type e The results database contains multiple records called results records Within each record is a set of fields containing informat
47. When calculations are complete the corrected absorbances are displayed under the Pathlength Correction results tab Typical results are shown on the next page User s Guide to KCjunior Interpreting Results e 7 17 Raw Data Pathlength Correction Transformation Results Template 0 800 1 441 1 993 1 683 1 400 2 174 1 428 2 846 i Wavelength m Pathlength Correction Options Corrected ODs Statistics C Pathlengths e The Corrected ODs are initially displayed e Click the Pathlengths radio button to view the pathlength value for each well e Each corrected well contains the result of the following formula A wavelength well pathlength of well The method used to calculate pathlengths depends on the Pathlength Correction parameters defined under the Read Method tab in the protocol See page 5 16 for more information e To view the corrected absorbances at a different wavelength choose a value from the Wavelength drop down menu e If Bio Tek s Bio Cell plate was used to calculate the absorbance of water at 1 cm value click the Bio Cell Results button to see the absorbance readings at the Test and Reference Wavelengths Bio Cell Results Dialog Ea Absorbance Test Wavelength 0 238 977 nm Reference Wavelength 0 056 900 nm Cancel Read Difference 0 1 82 7 18 e Interpreting Results User s Guide to KCjunior Transforma
48. With Plate Formulas To apply an existing Plate Formula 1 To the right of the Plate Formula field click the down arrow to see a list of available formulas 2 Click on the desired formula to select it gt During data reduction this formula is automatically applied to every well that has been assigned a Well ID To define and apply a new Plate Formula 1 Type the formula in the Plate Formula field See Formula Syntax Order of Precedence and Examples later in this section for more information Alternatively let KCjunior assist with defining a new Plate Formula gt Click the ey button next to the Plate Formula field to access the Formula dialog See To define and apply a new Transformation Formula under Working with Transformation Formulas for instructions Add to List 2 Optional Click the Add to List button to save the new Plate Formula User s Guide to KCjunior Defining Protocols e 5 49 CV 5 50 e Defining Protocols Formula Syntax In KCjunior a formula contains a combination of Functions Well IDs Mathematical values and symbols and Location indicators Formula elements are listed and described below The operator represents addition e PC NC calculates the sum of the PC mean and the NC mean e X 0 005 calculates the sum of the current well and 0 005 The operator can represent either subtraction or negation e PC BLK subtracts the BLK mean from the PC mean
49. attempted a Standard Deviation on a well group with an insufficient sample population v attempted a Coefficient of Variation on a well group with an insufficient sample population e The well is in a blank sector that contains blanks but none of them are valid e Cutoffs could not be evaluated for the well e A concentration had no valid solutions on the generated curve e The curve could not be generated indicates a well was set to the maximum valid value The Raw Data value measured for this well exceeded the upper limit of the valid data range that was defined in the protocol KCjunior is currently configured to use this altered value in further calculations Suggestions e To extend the upper limit of this range or to exclude this and all out of range wells from further calculations 1 Click 2 to access the General Information dialog 10 4 e Troubleshooting User s Guide to KCjunior User s Guide to KCjunior 2 Do one of the following Set the Raw Data or Blank Limits Maximum Value to the desired OD RFU or RLU Select Discard Out of Range Values for Raw Data Limits or Blank Limits as appropriate All out of range wells will be reported as OUT 3 Click OK to accept the change indicates a well was set to the minimum valid value The Raw Data value measured for this well fell below the lower limit of the valid data range that was defined in the protocol KCyjunior is currently configured to use
50. below Raw Data Template 0 857 0 875 0 889 0 900 1 409 1 447 1 450 1 990 1 991 1 991 2 017 2 019 2 021 2 022 2 024 2 037 2 038 2 039 2 040 1 771 1 771 1 772 1 772 1 775 1 776 1 776 1 776 1 778 2 833 2 835 2 837 2 850 2 852 2 853 2 855 Wavelength 405 Empty 7 405 Empty Statistics 405 Read 405 Result hb 630 Result e Each well contains the OD value measured at the wavelength displayed in the Wavelength field Use the drop down list to choose a different wavelength e Ifan empty plate was read at a particular wavelength results will be provided for the Empty plate the Read plate and the difference between the two Result fo E 7 Read Emply Plate The read method parameters shown to the left correspond with the results shown above 630 I Read Empty Plate Read Method parameters User s Guide to KCjunior Interpreting Results e 7 13 Raw Dispense Read Data Raw data results and kinetic curves are viewed under the Raw Data tab To switch between the different views click the appropriate Kinetic View Options radio button Individual Reads Raw Data results for individual kinetic reads are shown below Raw Data Template 2033 6045 11057 11069 21080 21081 21092 21093 31104 31105 Kinetic Read Kinetic View Options fi Paw Data Kinetic Curve Data
51. button was clicked to halt the read in progress e KCjunior will attempt to perform data reduction based on any data retrieved up to the time the button was clicked Reader control error occurred during read This warning will appear if during a plate read the reader experiences a failure and is unable to respond to the computer e The reader control error may appear if there is a serial communication failure during the plate read e If the reader control error appears try the following gt Turn off the reader gt Ensure that the serial cable is properly connected Troubleshooting e 10 9 Close KCjunior then reboot the computer Turn the reader on Run a system test Launch KCjunior VV V WV Under Setup Reader check the communication parameters then click Test Communications If the test is successful try re reading the plate e Refer to the reader s Operator s Manual for information on specific reader failures and corrective action Strikethrough indicates a masked well A well with a strikethrough 2 57 indicates that the well is masked or temporarily excluded from calculations To unmask the well 1 Highlight the well and click the right mouse button 2 Select Mask Well 3 KCjunior will automatically recalculate the results Stored curve does not exist The protocol references a stored curve that does not exist in the current Results Database Suggestions e Choose to modify the
52. faucet ctecteas cack dcns adiabasiacchated T R 4 Connecting the Reader to the Computer ssasj soc sacs caccasscarssotaseiassencsanueetessnsneuessatetdusaatsennssaisedes 4 2 Reader SG BU Pie neas tues taeasaaiatuesaactepaitics Sas zersatat austen au suiaatoiutenene caneaeoueeanomnaaualounuasncnenatem 4 3 SYS ENEC ONOM AMON isos E A 4 5 Well TG CH C0 aerea a O 4 5 Reporting OPONSE a satis aaa 4 7 General Detail t Informa oms snis tavedstelea te dd Hebeetonideeaddiawees 4 8 Eas 10120 6 xc mena NET ne eo ER ane RTE ae aa Rr ee ee eer ee ne 4 9 Filter Whee UC ar 8 6S 03 cai cica Scop cutccua esses added casrioeas somes cuban eaheinicead N 4 11 Defining Protocols 5 1 VI W aa le cal daintealie Me unhacatuagehie cal ten taaieet Uaunlaemt ideas hiecal oats 5 1 WOKE With PrOlLO CO lS ici oi casces E table sAiseuaaack dene cuaeea en ae N A 5 2 Where are Protocols Stord l asicac dan onionenn nee EE E SE 5 2 Opening an EP xistin Protocoles E tenet aencntaesaes 5 2 Creauine ad New Proto COl uesa is ance sateen esac Gide aside saiaesa esi ceoeueddaeencmds 5 3 INFOCITY na Protocol sna cuncauietec anus dwelads aodeatentwbenaatoc antes dumvinen tadentadewbieaptoiestdemeieanieds 5 4 Saving and Closing Protocols cccccsssssssseseeeeeceeeeeeeceeaaaaseeeeeeeeeeeeeeeeeeeeeeaasaeeesanes 5 4 COP VIS PROTOCOLS sns a a a e en Sianees 5 5 Sharning Protecols and Databases ai 24 uinchcesvivialecudonass Vandi Nady tai se auinaee 5 5 porine PLOTOCOIS 415 2 ceic
53. field is dimmed and inaccessible the Automatic Sensitivity Adjustment feature is enabled Click the Options button to specify Sampling parameters and or to enable the Automatic Sensitivity Adjustment feature See Specifying Filter Set Options for further information User s Guide to KCjunior Click the Options button to define Sampling and or Automatic Sensitivity options for a filter set 45720 a300 Bottom E 150 Options User s Guide to KCjunior Specifying Filter Set Options Each filter set has its own configuration of options for well sampling and for automatic sensitivity adjustment if required Filter Options Filter Set 1 Ea Sampling Samples per Well ji Cancel Delay Before Sampling msec Help Delay Between Samples msec ji Get Defaults Sensitivity Adjustment Automatic Sensitivity Adjustment f Scale to Low High Range Scale to High Well Low Well Range at Ad Base Value 100 Test Well Range 01 D2 High well Range H1 H High alue sq000 M Use this result for all filter sets with automatic sensitivity adjustment Sampling KCjunior provides three Sampling options to specify the well reading characteristics for a filter set Note Click the Get Defaults button to set all three options to their original default settings To define the Sampling options L Specify the Number of Samples measurements to take per well The valid entry range is
54. formula 5 51 Sampling 5 27 Selecting the set for curve calculations 5 51 5 56 Sensitivity 5 26 Filter wheel cartridges Configuration 4 11 User s Guide to KCjunior Emission 4 11 5 23 Excitation 4 11 5 23 Selecting at run time 6 17 Selecting within a protocol 5 23 Filters retrieving from reader 5 36 First Excel cell location for data export 6 18 Fluorescence Auto sensitivity results 7 32 Blanked Data 7 16 Chemistry 5 21 Dispense Read 5 10 5 21 Endpoint read 5 10 Kinetic read 5 10 Raw Data 7 4 Relative Fluorescence Unit RFU 12 1 FLx800 Configuring filter wheel cartridges 4 11 Filter set definition in protocol 5 24 Filter wheel cartridge selection in protocol 5 23 FLx800 dispenser Filter position requirements 4 14 Initializing 9 5 Injector position 9 7 Priming 9 6 Purging 9 6 Syringe setup 9 7 Formula Coefficient of variation CV 5 50 Copying from well to well 5 47 DIL function 5 51 Help with creating 5 47 5 49 5 60 Filter sets FS 5 51 MEAN function 5 51 Order of precedence 5 53 Plate formula 5 49 Saving 5 48 5 49 Standard deviation SD 5 52 Syntax rules 5 50 Transformation formula 5 47 Validating 5 48 5 49 Value of current well X 5 53 Viewing results 7 19 7 29 Wavelength WV 5 52 Well location reference 5 52 Well reference lt well D gt 5 53 FS filter set function 5 51 7 19 7 20 7 27 7 29 G Get basecode version 4 3 Get communication defaults 4 4
55. luminescence reads to 6 sets of raw data are presented depending on the number of filter sets specified in the protocol Filter Set e Fecitation 485 20 Initially the RFU RLU values for filter set 1 are presented Emission 530 20 Use the drop down menu at the bottom of the screen to choose a different filter set e For multi plate endpoint reads use the Plate Number selection box to choose a different plate Plate Number 7 4 e Interpreting Results User s Guide to KCjunior Filter Set Filter Set 2 Filter Set 1 Filter Set 2 User s Guide to KCjunior Raw and Blanked Kinetic Read Data Raw and blanked kinetic data are viewed under the Raw Data tab View options include sets of individual kinetic reads the kinetic curve for each well and the maximum slope for each well Kinetic View Options C Raw Data Kinetic Curve f Blanked Data C Masinum Slope To switch between the different views click the appropriate Kinetic View Options radio button Individual Kinetic Reads Raw Data results for individual kinetic reads are shown below Raw Data Template 0 750 0 875 1 464 1 469 1 996 1 996 2 065 2 066 2 072 1 785 1 787 1 787 1 789 1 789 2 921 2 922 Kinetic Read Kinetic View Options ONE Help pS aimee ____tee _ Raw Data C Kinetic Curve Statistics C Blanked Data C Maximum Slope Statisties Kinetic data is initially
56. no filter or plug For a FLx800 that is used for a variety of applications multiple Excitation and or Emission filter wheel cartridges may be prepared and installed as needed It is important for KCjunior to know the exact values and positions of the filters plugs holes for the cartridge currently in use To simplify the filter definition process when multiple Excitation and or Emission filter wheel cartridges are used the contents of each cartridge can be entered once into KCjunior and given a name such as Cartridge Excitation 01 When changing to a different cartridge simply select its assigned name in KCjunior s Current Reader Menu to prepare to create protocols and read plates User s Guide to KCjunior Setting Up the System e 4 11 4 12 e Setting Up the System To add modify or delete a filter wheel cartridge configuration select Setup Configuration Click the Filter Wheel Cartridges tab KCjunior Configuration x well IDs Reporting Options General Pathnames Filter Wheel Cartridges Cartridge Emission 01 E Help Cartridge Emission 02 Cartridge Excitation 01 Modify Cartridge Cartridge Excitation 02 Delete Cartridge m Filter Wheel Cartridge Prompt Option Prompt before every plate read Prompt if current cartridge selections do not contain required filters Never prompt e Click Add Cartridge to configure settings for a new filter wheel cartridge and add it to the list e Select a cartridge n
57. of Dispensers Supported ii Help Dispenser 1 Connected Initialize Dispenser Initialzed Prime Dispenser Frimed Purge Dispenser Injector Positions Set Injector Position Dispense Events Syringe Setup Sampling Windows Supported The maximum number of unique sample measurement scenarios supported per well per kinetic read in a Dispense Read protocol Number of Dispensers Supported The maximum number of dispensers that the reader currently supports For each dispenser the following information is provided Connected Indicates whether or not the reader recognizes that the dispenser is connected Initialized Indicates whether or not the dispenser is initialized and ready for use Primed Indicates whether or not the prime operation has been run since the dispenser was last initialized Injector Position Displays the reader s current injector position setting Top or Bottom Dispense Events The maximum number of dispenses supported per well in a Dispense Read protocol Initializing the Dispenser If the dispenser was connected before the reader was turned on or if a system test was run the dispenser should initialize automatically If for any reason the dispenser does not initialize automatically you can initialize it from KCjunior This may be necessary if the dispenser was connected to the reader after the reader was turned on or following the installation of a new syringe To
58. p 5 16 Defining the Spectral Scan Wavelength Range p 5 20 Defining the Number of Plates to be Read p 5 20 Selecting a Read Mode p 5 35 e The following topics are specific to fluorescence luminescence readers Choosing the Chemistry p 5 21 Specifying Dispense Read Options p 5 21 Selecting Filter Wheel Cartridges p 5 23 Defining Filter Sets p 5 24 Specifying Filter Set Options p 5 27 Ejecting the Plate Between Filter Sets p 5 30 e The following topics apply to most readers Defining Kinetic Parameters p 5 31 Selecting the Plate Geometry p 5 31 Setting a Time Delay Before Reading p 5 32 Monitoring Wells p 5 32 Defining Incubation Parameters p 5 34 Defining Shake Parameters p 5 34 Validating Read Method Parameters p 5 36 Retrieving Reader Wavelengths Filters p 5 36 Sending Reader Wavelengths p 5 36 Calibrating the Reader p 5 37 Calibrate Before First Read p 5 37 User s Guide to KCjunior Defining Protocols e 5 15 Dual wavelength readings significantly reduce optical interference caused by scratches or fingerprints on the microplate Instruct KCjunior to calculate and report the difference in measurements between an empty plate and the sample plate 5 16 e Defining Protocols Selecting Primary and Reference Wavelengths If a Primary Wavelength is specified alone the plate is read at a single wavelength If a Reference Wavelength is also sp
59. plate read actually begins Notes For MultiWavelength reads if Read Empty Plate is enabled the delay begins after the empty plate s are read For multiple plate Endpoint reads the delay occurs before each plate is read To set a Delay Before Read 1 Enter the time in seconds in the Delay Before Reading field Monitoring Wells If the currently selected reader supports well monitoring the Monitor Well option is available This feature enables the instrument to repeatedly read a single well or a group of wells and evaluate the measurements against pre defined criteria Once the criteria are met the plate read begins To define Monitor Well parameters 1 Check the Monitor Well box Wheaten a Pepe ene 2 Click the Monitor Well Parameters button The Monitor Well Parameters dialog appears 5 32 e Defining Protocols User s Guide to KCjunior KCjunior database versions 1 4 and greater support the Monitor Well feature From the main menu select Help About KCjunior to see the version numbers for the currently selected protocol and results databases User s Guide to KCjunior Monitor Well Parameters Dialog Fa Criteria Average of wells gt 0 200 OD Cancel Delay Between Reads 5 Seconds Help Select well s to monitor Clear Plate 3 Specify the monitoring Criteria gt Note For the EL311 312 340 readers the criteria is limited to the entry of a
60. printing reports for a multiple plate endpoint read the Print Endpoint Plates dialog appears Print Endpoint Plates Dialog Humber of Plates Cancel First Plate to Print Last Plate to Print gt The Number of Plates field contains the total number of plates that were read gt Adjust the First Plate to Print and Last Plate to Print values to preview a subset of the total number of plates gt Click OK to continue with the preview User s Guide to KCjunior User s Guide to KCjunior Print Setup To change the printer paper size or source or page orientation select Results Print Setup to access a standard Windows Print Setup dialog The Print Setup dialog appears Hame admz hpiii k Properties Status Ready Type HP Lasernet Ill Wher WEtntev05adme hpill Comment Print to file Print range Copies i All Number of copies li C 1 2 i iia from to Pl elf sll F Colate Selection f Cancel e To select a different printer click the Printer Name drop down arrow e To change print properties such as paper type and resolution click the Properties button e To select a different paper size click the Paper Size drop down arrow e To select a different paper source click the Paper Source drop down arrow e To select the paper orientation click the Portrait or Landscape radio button e Click OK to save the changes and return to the KCjunior main screen Printing Rep
61. programs can only be used by a single user You may transfer the programs from one computer to another provided that the programs are used with only one computer at a time You may not electronically transfer the programs from one computer to another You may not distribute copies of the programs or documentation without the prior written consent of Bio Tek Instruments Inc or as stipulated in a sub licensing agreement Network Use You have the non exclusive right to use the enclosed programs These programs can only be used on a single file server The number of concurrent users on the network at any time may not exceed the number of licensed copies of this software You may transfer the programs from one server to another provided that the programs are used with only one server at a time You may not distribute copies of the programs or documentation without the prior written consent of Bio Tek Instruments Inc or as stipulated in a sub licensing agreement You may not use copy modify loan lease or transfer the programs or documentation or any copy except as expressly provided in this Agreement COPYRIGHT The programs and the documentation are copyrighted You may not copy any program or the documentation except as for loading the program into the computer server as part of executing that program and for back up purposes respectively You must reproduce and include the copyright notice on the back up copy TERMS This license is effe
62. protocol If the protocol is open KCjunior will automatically turn on the reader s incubator when a plate read is initiated The plate read will begin when the incubation temperature reaches and stabilizes at the setpoint temperature To use the Incubation Control utility 1 Ensure that the reader is properly connected to the computer and that all communication parameters are set correctly 2 Select Utilities Incubation Control The Incubation Control dialog appears Incubation Dialog Temperature Control Incubator Cancel Set Point C ie Duration Indefinite Duration Seconds 3 To turn the incubator on and incubate a plate now gt Click the On radio button gt Enter the Set Point temperature The valid range for most readers is from 20 C or 4 C above room temperature whichever is greater to 50 C gt Deselect Indefinite duration gt Enter the incubation Duration in seconds User s Guide to KCjunior Incubate a plate later User s Guide to KCjunior gt gt Click Start The Read Plate dialog appears showing the Actual Temperature of the reader as it rises toward the setpoint Read Plate Dialog Waiting for temperature to reach setpoint Sa Override Incubation Setpoint 37 Actual Temperature 26 3 Click the Override button to begin the incubation before the setpoint temperature is reached Note The acceptable temperature range for plate reading for mo
63. sample groups as SMP01 SMP02 SMPO03 and so on Within the Sample ID template you can assign custom identifiers to sample groups L 4 Click on any well in Sample group number 1 SMPO1 will appear in the Sample ID field Overwrite SMP01 with any name in the Sample ID field then click the Apply button The ID will be assigned to every sample in group 1 Repeat for several more samples Click the Curve tab Define Curve Parameters KCjunior provides three methods for generating curves A standard curve can be generated based on the standard wells defined within the protocol A previously generated and stored curve can be referenced from within the protocol A titer curve can be generated using multiple dilutions of sample groups as concentrations Here you will define parameters to generate a point to point standard curve L 2 Select the Point to Point curve method Check Extrapolate to display extrapolated concentrations unknown values on the Y axis which are outside the range of the standards Leave all other parameters set to their default values Click the Cutoffs tab Getting Started e 3 17 Define Cutoffs User defined Cutoffs are used to classify results During data reduction results are evaluated against the cutoff criteria and each well is assigned a user defined Label Here you will create three cutoff zones L Ds 4 Enter STDO1
64. single value the dilution defined for the well in the Dilutions template e DIL returns the currently defined dilution value for the well such as 10 000 e DIL X multiplies the defined dilution value by the current value in the well For example with a quantitative assay the X value may represent the calculated concentration for the well e DIL is not valid within a Cutoff formula The FS function is available for fluorescence and luminescence protocols and is used to specify a particular filter set for use in the formula e FS 4 100 multiplies the result in each well of Filter Set 4 by 100 e FS 1 FS 2 divides the result in each well of Filter Set 1 by the result in the corresponding well of Filter Set 2 e To control the basis for standard curve and concentrations calculation in a multiple filter set read apply FS lt filter set numbers to every well in the Transformation Template KCjunior automatically uses the results calculated for that filter set when generating the curve and calculating concentrations e FS is not valid within a Cutoff formula The MEAN function calculates the average of the contents inside the parenthesis It supports up to five parameters each of which must equate to a single value e MEAN 7 16 4 returns 9 e MEAN PC NC returns the mean of the PC and the NC replicate groups If there is a single parameter inside the parenthesis then it is assumed to be a Well ID e MEA
65. specified appropriately Click the Calibrate Reader button from the Read Method dialog KCjunior attempts to communicate with the reader If communication is successful the reader performs calibration and then runs the system test When the system test is complete the results are displayed Click OK to close the results and return to the Read Method dialog Calibrate Before First Read Calibrate Before First Read For Spectral Scan reads the Calibrate Before First Read option is available User s Guide to KCjunior If this box is checked the reader calibrates at all of the wavelengths defined in the protocol just prior to plate scanning If this box is not checked the reader calibrates at only those wavelengths that have not yet been calibrated since the reader was turned on Defining Protocols e 5 37 Templates Within each KCjunior protocol four unique Templates can be designed to indicate location and related information for Well IDs Dilutions Transformations and Sample IDs e Well Identifiers play an important role in calculations such as blank subtraction curve generation and cutoff evaluation e Dilutions can be specified to indicate a reduction in the amount of concentration in solutions e Transformations are user definable formulas that can be applied to one or more wells e Sample IDs can be defined to distinctly identify sample wells To design templates 1 Choose to create or modify a proto
66. template The Raw Data Report however can be printed even if no well ID s are assigned User s Guide to KCjunior Reports KCjunior provides up to seven different reports depending on the current protocol settings The contents of some of these reports can be further customized for greater flexibility with data presentation To select the reports for use with a particular protocol 1 Choose to modify an existing protocol or create a new one 2 Select the Reports tab The Reports dialog appears General Information Read Method Template Curve Cutoffs Reports Report Banner fs ample Report Setup Help MV Protocol Report MV Show Template Show Formula MV Raw Data Report Raw Data C Blanked Data Maximum Slope Report MV Formula Report C Transform Results Plate Formula Results Both I Show Formula T Column Report SotbySampleID C SortbyLocation Down Sort by Location Across MV Matrix Report Options Selections Well IDs __Add gt Raw Data Transformations Transformations Std Conce Dilutions _ lt Remove Calculated Concs Calculated Concs MV Curve Report Cancel 3 Define a Report Banner using up to 32 alphanumeric characters This banner appears at the top of each printed report 4 Check the box or radio button next to every report and corresponding option s to be printed gt The Protocol Report lists many of the parameters defined in this protocol Click Show Template to include a r
67. the one currently in use 3 4 e Getting Started User s Guide to KCjunior Utilities Menu The Utilities Menu contains special tasks including reader diagnostics and database maintenance Setup Curent Reader View Help Incubation Control Carrier Out Carrier In Dispenser Control Panel Diagnostics Database Maintenance Create Archive Results Database Create Archive Protocol Database Archive Results Archive Protocols Delete Results Delete Protocols Archive Stored Curves Archive System Test Results Archive Universal Test Plate Results Delete Stored Curves Delete Stored Transformations Delete System Test Results Delete Universal Plate Test Results Incubation If the attached reader supports incubation turn the Control incubator on off and set an incubation duration Carrier Out In Open close door or extend retract carrier Dispenser Perform setup prime and purge tasks specific to Conirol Panel the FLx800 with the fluid injection capability Diagnostics Perform system and alignment accuracy tests on the attached reader reader dependent Database Create Results and Protocol Databases for Maintenance archiving Archive or delete results protocols stored curves and reader test results User s Guide to KCjunior Getting Started e 3 5 Setup Menu The Setup Menu provides access to the reader setup and system configuration features Reader 1 Reader 2
68. this altered value in further calculations Suggestions e To extend the lower limit of this range or to exclude this and all out of range wells from further calculations Click to access the General Information dialog 2 Do one of the following Set the Raw Data or Blank Limits Minimum Value to the desired OD RFU or RLU Select Discard Out of Range Values for Raw Data Limits or Blank Limits as appropriate All out of range wells will be reported as OUT 3 Click OK to accept the change indicates an extrapolated concentration The concentration for this well was calculated through extrapolation If Extrapolate is enabled in the protocol the curve is extended outside the total range of the standards in order to calculate a concentration for an OD RFU or RLU value beyond the highest and lowest standard values Suggestion e To disable Extrapolation 1 Click 2 to modify the protocol 2 Click the Curve tab 3 Deselect Extrapolate 4 Click OK to accept the change and recalculate the results Troubleshooting e 10 5 indicates ambiguous concentrations Two or more concentrations were calculated for the Y value in this well e KCjunior automatically reports the largest concentration indicates a well read error A well read error may be caused by one of the following e The reader determined that the OD RFU or RLU in this well was significantly out of range then set the error status befo
69. 0 1 312 17 955 STDO1 A2 0 000 1 325 15 460 AVE 1 319 16 707 STD02 Bl 25 000 1 287 22 377 STD02 B2 25 000 1 244 29 334 AVE 1 265 25 856 STDO3 Cl 50 000 1 036 60 627 STD03 C2 50 000 1 205 35 283 AVE 1 121 47 955 STD04 D1 100 000 53 598 7222 STD04 D2 100 000 0 781 107 652 AVE 2 189 107 652 STD05 El 200 000 0 527 192 560 STDO5 E2 200 000 0 526 193 073 AVE 0 526 192 816 STD06 Fl 400 000 0 322 414 352 STD06 F2 400 000 0 324 409 482 AVE 0 323 411 917 Controls Blanks Samples Well ID Sample ID Loc Dilution Raw Data Calc d Conc 0100 Gl 1 000 0 512 200 524 0100 G2 1 000 0 484 217 068 0 498 208 796 0200 H1 1 000 1 201 35 883 0200 H2 1 000 1 192 37 227 1 196 36 555 0300 A3 1 000 1 238 30 265 0300 A4 1 000 1 239 30 110 1 239 30 188 0400 B3 1 000 1 110 49 383 0400 B4 1 000 1 097 51 325 1 103 50 354 0500 C3 1 000 1 255 27 607 0500 C4 1 000 1 209 34 682 1 232 31 145 0600 D3 1 000 1 320 16 440 0600 D4 1 000 1 298 20 480 1 309 18 460 1 1 30 2000 9 04 40 AM Column Report page I of 2 User s Guide to KCjunior Plate Read Time 11 30 2000 8 23 59 AM Temperature n a Std Dev Label NEG NEG NEG NEG EQV EQV EQV EQV EQV 999999 EQV EQV POS EQV POS POS POS Std Dev Page 1 of 2 Appendix A Sample Reports e 11 7 COLUMN REPORT cont Protocol ID Sample Protocol 4 Plate Read Time 11 30 2000 8 23 59 AM Plate ID Sample Results 4 Temperature n a Well ID Sample ID Dilution Raw Data Cale d Cone
70. 05 25 PM 100414 aia 11 5 99 3 10 47 PM 100415 _ Hp Delete after archive Select the test result s to be archived gt To switch between ascending and descending sort order click the Test Results column header gt Use the mouse to select the items s to be archived Press the Shift key while using the mouse to select contiguous items Press the Ctrl key while using the mouse to select non contiguous items gt Check Delete after archive to permanently delete the item s from the current database after being copied to the archive database User s Guide to KCjunior User s Guide to KCjunior Click the Archive Test s button The selected items are copied to the Archive Results Database and then deleted from the current database if Delete after archive is checked gt Ifa duplicate Date Time is detected KCjunior does not overwrite the existing test results Deleting Results Records and Protocols After results records and protocols have been safely archived or if certain results records or protocols are no longer needed they can be deleted from the current Results and Protocol Databases To delete results records and protocols L 2 Close any open records protocols Select Utilities Database Maintenance Delete Results or Delete Protocols The Delete Results or Delete Protocols dialog appears listing all results records or protocols in the current database Delete Protocols Dialog Ea Protocol Nam
71. 1 Ensure that the Default Well IDs for Blank Standard Sample and Control wells are defined appropriately under Setup Configuration 2 Ensure that the correct Plate Geometry is selected under the Read Method tab 3 From within the Well ID Template select a well type from the Well Type Selections drop down menu gt The Well Type Selection names are the descriptive links between the generic well type 1 e Blank Sample Standard and Control and the 2 to 5 character Well ID Well Type Selection names and Well IDs are configured under Setup Configuration Well IDs Defining Protocols e 5 39 5 40 e Defining Protocols If applicable define the Number of Replicates Replicate Direction and Map Direction associated with that ID gt Specify the Number of Replicates for the selected well type The Number of Replicates value represents the number of individual replicates 1 2 3 within a single group SMP BLK STD KCjunior automatically increments the numeric ID suffix gt Specify the Replicate Direction Down or Across The Replicate Direction is the direction in which individual replicates within a single group are mapped A direction of Down maps replicates down columns e g A1 B1 C1 D1 A direction of Across maps replicates across rows e g Al A2 A3 A4 gt Specify the Map Direction Down or Across The Map Direction is the direction in which the groups are mapped If applicable enter the Standard or
72. 1 to 255 measurements The value reported for each well represents the average of its measurements gt The reader spends approximately 10 milliseconds performing each measurement gt Usually the more samples per well the better the CV s although selecting more than 10 samples per well typically results in only marginal improvement 10 is the recommended combination of sensitivity and speed Defining Protocols e 5 27 The Help system shows how to define pre reading para meters for a sample assay 1 Click the Help button from within the Filter Options dialog 2 Click on the last option Examples 5 28 e Defining Protocols 2 Set the Delay Before Sampling in milliseconds This is the pause time to allow plate contents to settle before the measurements begin The valid entry range is 10 to 2550 milliseconds in 10 ms increments gt 350 milliseconds is the recommended setting to ensure the plate is definitely stopped for measurement and for the minimum amount of time 3 Set the Delay Between Samples in milliseconds This is the time delay between samples The valid range is O to 255 milliseconds gt For fluorescence assays a delay of 10 milliseconds is recommended Note Delays longer than 200 milliseconds may bleach the samples gt For luminescence assays longer delays between samples may result in better CV s Automatic Sensitivity Adjustment KCjunior provides an Automatic S
73. 13 0 819 0 875 1 058 1 305 Transform nooo ioo oao isoo 128o sozod 65 700 st 300 isoo 87300 105300 130300 Pi Trams 0 838 0 7 0 944 0 88 0 925 25 0 68 0 727 0 646 0 828 0 816 0 938 Transform sa nm oaao ssam osoo 105704 eemo 2o sso s20 siso osso Pi Tran 760 45 0 890 1 069 1 656 398 0 790 1 137 1 82 1 825 1 85 1 067 Transform amoo 245200 sooo 106900 165 609 239800 mooo 137o 182200 192 50 185100 106 700 rit Trams g 1 707 1 638 1 074 1 828 1 581 0 949 0 901 0 801 2 147 2 101 518 1 775 Transform mso isoo orao isso Isso 9490q ooroo soioo 214700 210100 251800 177 500 Pit Trans Transformations Transformation Formula A 2 A 12 2 A 12 B 2 B 12 2 B 12 C 2 C 12 2 C 12 D 2 D 12 2 D 12 E 2 E 12 2 E 12 F 2 F 12 2 F 12 G 2 G 12 2 G 12 H 2 H 12 2 H 12 Plate Formula X 100 04 23 99 09 15 20 AM Page of 1 Formula Report with Transformation and Plate Formula information 11 6 e Appendix A Sample Reports User s Guide to KCjunior Sample Report COLUMN REPORT Protocol ID Sample Protocol 4 Plate ID Sample Results 4 Warning Messages OUT indicates a well exceeded the min max range 27727 indicates a calculation could not be performed An S in front of a value indicates a suppressed standard indicates an extrapolated concentration Standards Well ID Loc Concentration Raw Data Cale d Conc STDO1 Al 0 00
74. 36 74 052 24 684 63 474 23 385 55 539 22 216 49 368 4 232 44 431 4 039 40 392 3 864 3 555 32 197 3 418 31 512 3 174 30 225 3 064 4 29 621 2 962 29 040 37 026 28 482 36 123 27 944 35 263 27 427 34 443 26 928 33 660 26 447 A 25 535 F 12 009 25 103 11 848 13 671 11 541 13 464 4 11 393 i 13 263 11 248 13 068 11 108 12 879 10 971 12 695 10 837 12 516 10 706 12 342 10 579 71 090 es 10 333 10 214 70 386 9 985 69 696 69 020 Kinetic View Options Raw Data Blanked Data 9 874 68 356 9 765 67 705 9 659 67 066 C Kinetic Curve Maximum Slope 9 555 66 440 74 052 65 824 73 289 65 220 Calculation Options 72 541 64 628 Help Statistics e The Maximum Slope is initially based on the total number of kinetic points single slope e If blank subtraction is performed the maximum slope is automatically based on the Blanked values e Click the Calculation Options button to adjust the kinetic read limits to alter the maximum slope calculation or to choose a different maximum slope unit of measure User s Guide to KCjunior Interpreting Results e 7 7 Adjusting Kinetic Read Limits Kinetic calculations initially include all measurements taken over the duration of the plate read Kinetic read l
75. 4 0 8986 B 2 2718 C 47 4425 D 23 9208 4 364 e The curve fitting method chosen in the protocol and its equation are displayed under Curve Type e If defined in the protocol the Interpolation Value is provided e Each Sample ID is listed in alphabetical order e For each sample the Curve Parameters and corresponding R Square values are reported e Ifan interpolation value was defined its calculated value for each titer curve is reported in the Interpolation column Interpreting Results e 7 23 7 24 e Interpreting Results Interacting with the Graph The displayed graph can be scaled moved and zoomed and the altered display can then be printed or copied to the Clipboard The graph report name and and curve equation information does not scale move or zoom with the graph Note At any time press r to reset the graph to its original size and position To scale the graph 1 Press and hold the Ctrl key 2 Press and hold both mouse buttons or the middle button of a 3 button mouse 3 Move the mouse down to increase the graph size or up to decrease the graph size To move the graph 1 Press and hold the Shift key 2 Press and hold both mouse buttons or the middle button of a 3 button mouse 3 Move the mouse to change the position of the graph To zoom in on a portion of the graph 1 Press and hold the Ctrl key 2 Press and hold the left mouse button 3 Drag the mouse to select the zoom area 4
76. 5 Cutoffs 3 11 5 58 Statistics 7 30 Transformations 3 11 5 46 Unit of measure 7 8 7 9 MEAN function 5 51 Microsoft Excel exporting plate data to 6 18 Microsoft Windows operating system 2 2 Minimum and maximum raw and blanked data values 5 8 5 9 Monitoring wells Defining the criteria 5 33 Elapsed time 7 35 Selecting the well s to monitor 5 34 Multiple plate endpoint read 5 20 6 10 Multiple wavelengths Empty plate 5 16 Pathlength correction 3 10 5 17 Referencing in a formula WV 5 52 Selecting 5 16 Selecting the set for curve calculation 5 52 5 55 Viewing results 7 4 7 5 Multiplication in a formula 5 50 MultiWavelength read 5 10 12 1 User s Guide to KCjunior N Network sharing databases 5 5 6 5 Number of plates 5 10 5 20 Number of samples 5 27 O OD 5 9 12 1 OMNI System absorbance files opening 6 3 6 13 Opening a results record 6 2 Optical density 5 9 12 1 Optics position 5 25 Optimum sensitivity 5 26 5 28 OUT indication 6 15 7 2 7 37 10 5 Outliers editing 7 21 7 25 Output empty row between plates 6 18 Output text headers 6 18 Over indication 7 37 P Parenthesis using in a formula 5 50 Parity 4 4 Partial plate reading 5 32 Password obtaining from Bio Tek 2 16 2 17 Pathlength correction 3 10 5 17 7 17 Pathnames 4 5 4 10 4 12 5 2 5 6 Peak OD 7 11 Peak wavelength 9 12 Peak wavelength test 9 10 Plate Closing 6 4 Copying 6 4 Description 6 8 6 10 6 12 6 13 7 35 Expor
77. 7 6 18 e Reading Plates Exporting Data Plate data can be exported to a file or directly into a Microsoft Excel spreadsheet The data options available for export depend on the current protocol parameters For example if the protocol performs blanking uses transformations and generates a standard curve the raw data blanked data transformation results and or calculated concentrations can be exported Note If no protocol is open the raw data can still be exported To export data to a file or to an Excel spreadsheet 1 Open Create a protocol if desired then read open a plate 2 Select Results Export Data The Export Data dialog appears Export Destination File Directly to Microsoft Excel Data Options Selections Raw Data Add Raw Data Transformations d gt Transformations Std Conc Dilutions Calculated Cones Calculated Cones lt Remove Well IDs I Output report header MV Qutput data descriptions IV Output empty row between plates Export File C KCjunior Cytotoxicity Il tet Browse Delimiter Tab Comma Other ji gt Review the items under Essential Exporting Information on page 6 23 before performing a data export 3 From within the Export Data dialog choose an Export Destination gt Choose File to export the data to a text file The Export File and Delimiter options are presented See Exporting Data to a File on page 6 19 gt Choose Dir
78. 79 nes Blkd Date H 1 746 1 638 1 072 1 824 1 581 0 947 897 0 798 2 143 2 085 2 499 1 764 Raw Data 1 283 1 175 0 609 1 361 1 117 0 484 0 434 0 335 1 680 1 621 2 035 1 301 Blkd Data 11 18 99 4 10 11 PM Page 1 of I Raw Data Report 11 4 e Appendix A Sample Reports User s Guide to KCjunior Sample Report 6x8 well format MAXIMUM SLOPE REPORT Protocol ID Sample Protocol 3 Plate Read Time 04 22 99 03 22 28 PM Plate ID Sample Plate 3 Temperature n a l J s o E ee ee ee ee eee eae Ee ee a ee ees Bal 3646 15 2884 62 4392 31 3807 69 4015 38 3461 54 3461 54 ee 4500 00 000 00 Max Slope 4546 15 07 69 07 69 046 15 861 54 769 0 77 Max Slope ae 2630 77 3253 85 2838 46 4015 38 3392 31 3461 54 3923 08 3461 ad ae 04 23 99 09 13 57 AM l Page l of 1 Maximum Slope Report 6x8 Plate Geometry User s Guide to KCjunior Appendix A Sample Reports e 11 5 Sample Report FORMULA REPORT Protocol ID Sample Protocol 1 Plate Read Time 04 23 99 08 47 27 AM Plate ID Sample Plate 1 Temperature 37 0 C O SN NS Js ERE E S 21 0 474 0 668 a 5 a s 0 636 0 924 0 70 a a orm B 0 949 0 675 0 995 0 902 1 O71 T 747 1 70 1 864 0 786 l 628 0 G90 l 23 Transform ee ae ee a E 7 146 7 860 424 5500 o 7 50 04 oF 9 _ranstorm o so mand eso seod iao sosoo 1orsod szi oio 8640 89500 Pu Trams O85 40 0 790 758 048 0 741 0 8 0 781 T 06 1055 7 980 09 0 Transform T33 1 320 0 894 1 760 1 I T 802 0 857 7 8
79. 8 1999 2000 All Rights Reserved 2 Write down all of the version numbers and the serial number 123456 in the example above 3 Click the Reader Control Version button A dialog resembling the following appears About BTlAutoreader Control m Bio T ek Autoreader Control Yersion 1 30 1 Fart Humber 5270203 Copyright Bio Tek Instruments Inc 1998 1999 2000 All Rights Reserved Write down the BTI Autoreader Control version Prepare a list of specific steps that can be used by Bio Tek s Technical Assistance Center TAC to reproduce the problem User s Guide to KCjunior Introduction e 1 3 www biotek com 1 4 e Introduction 6 Contact Bio Tek Instruments Be sure to provide a daytime phone or fax number or email address where you can be reached Written Communication Bio Tek Instruments Inc Technical Assistance Center Highland Park Box 998 Winooski Vermont USA 05404 0998 Phone Support Bio Tek Instruments Main Number 802 655 4040 Technical Assistance Center 8 30 am to 5 30 pm EST 800 242 4685 Fax Support Technical Assistance Center 802 655 3399 Electronic Communication Email labtac biotek com Internet Site www biotek com Visiting Bio Tek s Website If you have an Internet browser installed on your computer you can visit Bio Tek s World Wide Website at www biotek com Here you can find information on hardware and software products read application notes and m
80. Comparison Value OD When the selected well reaches the defined comparison value monitoring stops and the plate read begins From the first drop down list select a statistic of At Least One Well Average of wells or All Wells Note If only one well will be monitored select any statistic The behavior will be the same for all three options From the second drop down list select a logical operator lt gs gt 01 gt Enter a limit value This is the target measurement value for the selected well s The valid range is reader dependent Examples At Least One Well gt 0 050 OD The plate read will begin when at least one of the selected wells is greater than or equal to 0 050 OD Average of Wells gt 0 300 OD The plate read will begin when the average value of the selected wells is greater than 0 300 OD All Wells lt 80000 LFU The plate read will begin when all of the selected wells measure less than 80000 LFU 4 Optional Enter a Delay Between Reads in seconds This is the delay between readings of the selected well s gt 0 indicates no delay Defining Protocols e 5 33 5 34 e Defining Protocols ay Highlight up to 8 wells to be monitored gt Ifa well is shaded it has been selected and will be monitored gt Any well s on the plate can be monitored even if the protocol specifies only a partial plate read gt Click the Clear Plate button to clear the currently selected wells gt
81. Conc x dilution 5 51 Concentrations Calculated concentrations 7 20 Defining expected 5 42 Viewing results 7 20 Configuring setup parameters 4 5 5 39 Configuring well identifiers 4 7 Connecting the reader to the computer 4 2 Constant value 5 18 Contacting Bio Tek Instruments Inc 1 3 Continuous shake 5 35 Index e 13 1 Control panel dispenser 9 4 Controls defining 4 6 5 38 Copying a results record 6 4 Copying formulas 5 47 Copying plate data to another application 6 24 Corrected absorbance 5 18 Create archive database 9 16 Cubic curve 5 56 Cubic spline curve 5 56 Current reader menu 3 6 4 2 Current reader status 3 6 Curve Average Standards 5 57 Curve Method 5 56 Editing parameters 7 21 Extrapolate 5 57 7 2 Graph Report Name 5 56 7 21 Outliers 7 25 Overlaying 7 22 Printing 5 63 7 25 Report 5 61 Sample curves 7 22 Spectral scan 7 11 Standard 5 55 7 21 Stored 5 55 Titer 5 55 7 22 X and Y Axes Label Scale 5 57 Cutoffs Analyzing results 7 27 Defining 5 58 Labels 5 58 7 27 CV function 5 50 12 1 D Data bits 4 3 4 4 Data reduction 3 10 7 1 7 29 Errors 10 5 10 7 10 12 Database 4 9 5 2 5 5 6 2 6 5 12 1 Database maintenance A note on backup procedures 9 14 Archiving results records and protocols 9 15 Archiving stored curves 9 17 Archiving test results 9 18 Creating archive databases 9 15 Deleting results records and protocols 9 19 Deleting stored curves 9 20 Deleting stored transfo
82. Configuration Reader 1 or 2 Define communication settings for the reader s attached to the computer Configuration Define various default configuration parameters including Well IDs Reporting Options Pathnames and Filter Wheel Cartridges Current Reader Menu If two readers are connected to the computer use the Current Reader Menu to switch between them before working with protocols or reading plates eee View Help w FLs800 Corn UL Want Corn Select Filter Wheel Cartridges Get Current Reader Status Reader 1 Designate the first reader as the Current Reader Reader 2 Designate the second reader as the Current Reader Select Filter This option is only available if a fluorescence Wheel luminescence reader like the FLx800 is defined Cartridges as Reader or Reader 2 Use it to choose from a predefined set of filter wheel cartridges Get Current Retrieve the current status of the attached reader Reader Status For example if the reader encounters an error select Get Current Reader Status to retrieve the error code 3 6 e Getting Started User s Guide to KCjunior View Menu The View Menu provides options for controlling the amount of information displayed within the main screen w Toolbar w Status Bar Toolbar Show or hide the toolbar at the top of the screen The default is show Status Bar Show or hide the status bar at the bottom of the screen The default is show Help Menu The H
83. ID list box The Universal Plate ID list box should contain a list of data sheets associated with the universal test plate There should be one data sheet for each wavelength used for the test Universal Plate Test Dialog Universal Plate 1D wavelength 129375 450 14129975 6301 Operator k Eritting New Data Sheet i Yiew r Modify Data Reader Serial Humber 100413 Delete Data Sheet Comments Echeduled maintenance I Perform Peak Wavelength Test gt If all of the required data sheets exist for the test plate highlight the first Plate ID Wavelength to test then skip ahead to step 5 gt If one or more data sheets need to be created click the New Data Sheet button See Setting Up a New Data Sheet for more information User s Guide to KCjunior gt If one or more data sheets needs to be viewed or modified highlight a data sheet and click the View Modify button gt To delete a data sheet highlight the desired item in the Universal Plate ID list then click Delete Data Sheet KCjunior will request confirmation before deletion If necessary modify the Operator name 6 Enter the Reader Serial Number and any Comments If supported by the reader Perform Peak Wavelength Test is accessible If this box is checked the Peak Wavelength Test will be included in the universal plate test 8 Click the Run Test button The reader performs the test and KCjunior displays the results 9 Clic
84. N BLK returns the mean of the BLK wells Defining Protocols e 5 51 SD WV 5 52 e Defining Protocols The SD function calculates the standard deviation of the contents inside the parenthesis The Standard Deviation is a measure of the distribution of individual values relative to the mean The SD function supports up to five parameters each of which must equate to a single value e SD 3 124 3 094 2 997 returns 0 06638 e SD CTRL1 CTRL2 returns the standard deviation for all replicates of both control groups If there is a single parameter inside the parenthesis then it is assumed to be a Well ID e SD NC returns the standard deviation of the group NC The WV function references the set of data at the wavelength specified inside the parenthesis e WV 450 references the set of data read at or calculated for 450 nm In a MultiWavelength read if an empty plate is read at 450 nm WV 450 represents the Result or the difference in readings between the actual microplate and the empty plate e WV 405 WV 630 in a well divides its 405 nm result by its 630 nm result e To control the basis for standard curve and concentrations calculation in a MultiWavelength read apply WV lt wavelength gt to every well in the Transformation Template KCjunior automatically uses the measurements at that wavelength when generating the curve and calculating concentrations e WV is not valid within a Cutoff formula
85. P03 G9 8 000 103 728 6 974 SMP03 G10 8 000 104 052 6 530 SMP03 Gl1l 8 000 105 457 4 855 SMP03 H9 4 000 110 427 1 405 SMP03 HIO 4 000 105 240 5 088 SMP03 Hil 4 000 107 509 3 033 AVE 91 415 53 231 163 805 133 314 Titer Results Information Curve Type 4 Parameter y A D 1 x C B D Interpolation Value 50 000 Specimen ID Curve Parameters R Square Interpolation SMPO A 112 6209 B 1 0524 C 43 2727 D 0 9677 0 9947 54 596 SMP02 A 121 0374 B 0 6725 C 150 6673 D 28 6119 0 9986 129 595 SMP03 A 123 2737 B 0 2989 C 2239 4425 D 6 0131 0 9800 5500 44 11 30 2000 9 33 26 AM Page 4 of 4 Column Report showing Titer Results Information User s Guide to KCjunior Appendix A Sample Reports e 11 9 Sample Report MATRIX REPORT Protocol ID Sample Protocol 6 Plate Read Time 06 23 98 01 10 36 PM Plate ID Sample Plate 6 Temperature n a Warning Messages OUT indicates a well exceeded OD range An S in front of a value indicates a suppressed standard indicates an extrapolated concentration 04 23 99 12 59 46 PM Page 1 of 1 Matrix Report for an absorbance assay 11 10 e Appendix A Sample Reports User s Guide to KCjunior MATRIX REPORT Protocol ID Sample Fluoro 2 Plate Read Time 11 18 99 4 13 53 PM Plate ID Sample Fluoro 2 Temperature n a 98568 10078 94950 84872 0 58450 0 58129 0 55635 0 11 18 99 4 29 46 PM Page of I Matrix Report for a fluorescence assay Use
86. PO2 eM Fl 2 SMPO1 SMPO1 SMPO1 SMPO2 SMPO2 SMP02 4 BLE11 100 4 BLE SMPO1 Transformation Formula K BLK1 CTL1 BLK1 100 a Apply to Selection Apply To All Wells Options Add to List Help A e Add to List a concet_ gt Use this template to define modify and apply Transformation and Plate Formulas Plate Formula User s Guide to KCjunior Appl To All Wells Apply to Selection See Formula Syntax for information on mathematical values and symbols and location indicators User s Guide to KCjunior Working with Transformation Formulas To apply an existing Transformation Formula to one or more wells L To the right of the Transformation Formula field click the down arrow to see a list of available formulas Click on the desired formula to select it To apply the formula to all wells click the Apply To All Wells button To apply the formula to specific wells use the mouse to highlight one well or a block of wells then click the Apply to Selection button To access Copy Paste and Undo features click either the right mouse button or the Options button To define and apply a new Transformation Formula L Type the formula in the Transformation Formula field See Formula Syntax at the end of this section for more information Alternatively let KCjunior assist with defining a new Transformation Formula
87. Sample Number to be associated with the starting replicate group For example gt Setting Standard Number to 1 labels the starting standard group ID 01 such as STDO1 gt Setting Sample Number to 6 labels the starting sample group ID 06 such as SMP06 For well types of Standard and Sample check Auto Increment to automatically increment each subsequent group number by one After selecting the Well Type and defining any additional information assign the Well ID to one or more wells in the template gt To assign an ID to a single well click once on the desired well gt To assign an ID to multiple contiguous wells move the mouse cursor to the starting well then click and hold the left mouse button Drag the mouse cursor across and or down to the ending well then release the mouse button gt If the plate contains more than 8 rows and or 12 columns the template is viewable in quadrants To select a quadrant for well ID assignment click on the appropriate Quadrant number User s Guide to KCjunior 8 Ifthe template contains Blank wells select a Blanking Method Available methods are Full Half Quarter Row Column and Constant If Constant is selected set a Blank Constant value The valid Blank Constant range is 9 9999e 99 to 9 9999e 99 The Blanking Methods are described below Full Half Quarter Row Column Constant The average of the blank w
88. Std Dev CV Label 0700 E3 1 000 0 141 22277 27200 0700 E4 1 000 0 275 588 724 AVE 0 208 588 724 0800 F3 1 000 0 280 561 934 0800 F4 1 000 OUT AVE 0 280 561 934 0900 G3 1 000 1 117 48 341 0900 G4 1 000 1 076 54 487 AVE 1 097 51 414 1000 H3 1 000 0 461 232 656 1000 H4 1 000 0 432 255 652 AVE 0 447 244 154 Standard Curve Information Curve Type 4 Parameter y A D 1 x B D Parameters A 1 3753 B 1 6163 C 105 3927 D 0 2068 R Square 0 9862 Interpolation Value 1 000 Interpolation Result 66 327 11 30 2000 9 04 40 AM Page 2 of 2 Column Report page 2 of 2 11 8 e Appendix A Sample Reports User s Guide to KCjunior COLUMN REPORT cont Protocol ID SAMPLE Cytotoxicity Plate Read Time 1 9 1992 12 07 59 PM Plate ID SAMPLE Cytotoxicity Temperature n a Well ID Sample ID Loc Dilution Transform Cale d Conc Std Dev CV Label SMP03 A9 512 000 72 934 497 051 SMP03 A10 512 000 71 313 592 347 SMP03 All 512 000 74 878 401 605 SMP03 B9 256 000 80 173 220 477 SMP03 B10 256 000 81 037 199 265 SMP03 256 000 78 984 252 965 SMP03 C9 128 000 84 819 126 290 SMP03 C10 128 000 84 927 124 610 SMP03 Cll 128 000 84 495 131 449 SMP03 D9 64 000 91 194 54 889 SMP03 DIO 64 000 89 897 65 548 SMP03 D11 64 000 88 601 77 930 SMP03 E9 32 000 95 300 30 228 SMP03 E10 32 000 93 355 40 385 SMP03 Ell 32 000 93 247 41 024 SMP03 F9 16 000 96 488 25 141 SMP03 F10 16 000 97 569 21 151 SMP03 F11 16 000 98 325 18 679 SM
89. To define Kinetic parameters Select the Kinetic Read Method Type Enter the number of Kinetic Reads The Kinetic Read value represents the number of times the entire plate will be read L 2 Enter the Kinetic Interval in seconds The Kinetic Interval is the time delay between Kinetic Reads gt Alternatively allow KCjunior to select the minimum kinetic interval Check the Fastest Interval box to instruct KCjunior to calculate the minimum kinetic interval appropriate for the currently selected reader type This helps to ensure the fastest plate read possible After the Kinetic Reads and Kinetic Interval have been entered KCjunior automatically calculates the approximate Total Read Time and displays it in the HH MM SS format For the ELx800 PowerWave PowerWaveX WQuant and FLx800 readers gt When Fastest Interval is selected KCjunior calculates a theoretical minimum kinetic interval gt If the reader is connected to the computer click the Validate Read Method button to verify this value If the KCjunior theoretical value is too low the reader returns a valid range for the Kinetic Interval field gt If the reader is not currently connected to the computer and Fastest Interval is selected the Kinetic Interval is verified just prior to a plate read If the value is too low the reader returns a valid range for the Kinetic Interval field Selecting the Plate Geometry The Plate Geometry represents the number o
90. a range defined in the protocol Click 2 to open the General Information dialog to see the current Raw Data Limits and Blank Data minimum and maximum settings User s Guide to KCjunior User s Guide to KCjunior OUT indicates a well exceeded the min max range The Raw Data value measured for this well fell outside the valid data range as determined by the reader or as defined in the protocol Out of range values are currently being excluded from further calculations Suggestion e To include out of range wells in further calculations 1 Ifa protocol is open click to access the General Information dialog If no protocol is open select Protocol New Protocol The General Information dialog will appear 2 Alter the Raw Data Limits and or Blank Limits Minimum and Maximum values if necessary 3 Deselect Discard Out of Range Values for Raw Data Limits or Blank Limits as appropriate 4 Click OK to accept the change s All wells that were previously reported as OUT will be set to the minimum maximum values as appropriate and displayed in brackets These altered values will now be used in further calculations The Pathlengths could not be generated This warning will appear if during the read the Abort Read button was clicked to halt the read in progress before the necessary pathlength correction wavelengths were read Plate was aborted during read This warning will appear if during a read the Abort Read
91. a Plate With a Protocol To read a microplate with a protocol L Ensure that the reader is properly attached to the computer and that the correct reader type and communication parameters are selected under Setup Reader Open an existing protocol or define a new one See either Opening an Existing Protocol or Defining Protocol Parameters in Defining Protocols for more information Initiate the read by clicking on the Read Plate button in the KCjunior main screen selecting Protocol Read Plate or by clicking the toolbar icon The Read Plate dialog appears Read Plate Dialog Reader Jug uant Protocol Hame Dligonucleatide Results ID Simulation Data Read From File Cancel Help Dligoruclectide Plate Description LOT 765b Enter a Results ID using up to 32 alphanumeric characters The ID is optional in this dialog but it will be required later if the results are to be stored in the Results Database gt If performing a multiple plate endpoint read all of the plates read will be stored in the Results Database under this ID although they can each have their own Plate ID Enter a Plate Description using up to 1024 alphanumeric characters This description is optional User s Guide to KCjunior 6 Click the Read Plate button A second Read Plate dialog appears Read Plate Dialog E Place the plate on the carrier Press OK when ready Cancel Plate ID ooon a
92. able wavelength range is limited to 340 800 nm Defining Protocols e 5 3 Modify Protocol 2 5 4 e Defining Protocols Modifying Protocols To modify an existing protocol begin by opening the desired protocol select Protocols Open Protocol Once the protocol is open click the Modify Protocol button in the KCjunior main screen select Modify Current Protocol from the Protocol Menu or click the toolbar icon The General Information dialog appears containing identification information for that protocol See General Information at the start of Defining Protocol Parameters for more information Saving and Closing Protocols After creating or modifying a protocol save the information by selecting Save Protocol from the Protocol Menu or by clicking the toolbar icon e KCjunior stores the protocol in the current protocol database under the name defined in the General Information dialog To close an open protocol select Close Protocol from the Protocol Menu or click the toolbar icon If the protocol is newly created or if modifications were made to an existing protocol KCjunior provides an option to save KCjunior N Save protocol SAMPLE Cytotoscity e Click Yes to save the new or modified information and close the protocol e Click No to close the protocol without saving changes e Click Cancel to close this dialog and return to the open protocol The new or modified information is retained but not yet
93. ad 1 00 00 00 500 0 667 0 750 0 800 0 833 0 857 0 875 429 1 435 1 438 1 421 1 434 1 497 1 450 992r eI Sy Be OS a Foy Rae Oe Oh 209 029 2 031 2 032 2 033 2 035 2 036 2 037 empty row here Read 2 00 10 00 z465 1 55771 79371 510 1 835 1 987 1 675 429 2 433 2 438 2 441 2 484 2 447 2 450 942 2 895 2 045 2 963 2 223 3 017 3 019 029 3 031 3 032 3 033 3 035 3 036 3 037 Reading Plates e 6 23 Viewing Results When plate reading is complete or after simulation data has been entered or data has been read from a file KCjunior displays the results in a format similar to the one shown below Raw Data Blanked Data Pathlength Correction Transformation Results Concentrations Labels Template Plate Formula Results Comments Warnings 0 515 0 490 0 360 0 746 1 847 1 791 0 851 1 009 0 904 0 934 0 358 0 361 0 200 0 210 0 167 Initially Raw Data results are displayed Depending on the protocol definition KCjunior may also display Blanked Data Pathlength Correction Transformation Results Concentrations Labels Template and Plate Formula Results Some quick tips are listed here Detailed information can be found in the next section Interpreting Results e To view a particular type of data click on its tab e To view statistical information for a particular type of data click on its tab then click the Statistics button e To mask a well temporarily
94. adings at 977 and 900 nm are 0 175 and 0 025 respectively The pathlength for A1 is calculated as 0 175 0 025 0 18 or 0 8333 The sample plate is then read at 405 nm and the empty plate values are subtracted to determine the 405 Result values The 405 Result value for well Al is 2 576 The corrected value equals 2 576 0 8333 or 3 091 Example 2 A protocol specifies the following gt gt gt gt gt MultiWavelength read method type Microplate read at 405 nm Do not pre read an empty plate Pathlength Correction and Use Bio Cell are enabled Blank wells are defined in the well ID template The Bio Cell plate is read at the Test and Reference Wavelengths The absorbance of water at 1 cm 977 900 nm value is 0 182 The sample plate is initially read at the Test and Reference Wavelengths Well H1 readings at 977 and 900 nm are 0 208 and 0 034 respectively The pathlength for H1 is calculated as 0 208 0 034 0 182 or 0 956 The sample plate is then read at 405 nm and after blank well subtraction the 405 Result value for well H1 is 2 619 The corrected value equals 2 619 0 956 or 2 740 Defining Protocols e 5 19 Tip For a quantitative endpoint assay that calculates results based on a stored curve use the Number of Plates feature to read up to 100 plates 5 20 e Defining Protocols Defining the Spectral Scan Wavelength Range During a Spectral Scan the reader takes multiple measurements across a w
95. ame then click Modify Cartridge to modify settings for an existing filter wheel cartridge e Select a cartridge name then click Delete Cartridge to permanently delete the cartridge from the list e KCyjunior can be configured to present a prompt just prior to a plate read to allow the operator to specify which filter cartridges are installed on the instrument When a new cartridge is selected KCjunior updates the instrument with the new filter information gt Choose Prompt before every plate read to present the prompt before every FLx800 plate read gt Choose Prompt if current cartridge selections do not contain required filters to present the prompt only if there is a mismatch between the protocol and the instrument s filter table gt Choose Never prompt to prevent the prompt from appearing Note If there is a mismatch between the protocol and the instrument s filter table KCjunior presents an appropriate message User s Guide to KCjunior Adding or Modifying a Filter Wheel Cartridge From within the Filter Wheel Cartridges tab click Add Cartridge to configure a new cartridge or select an existing cartridge name and click Modify Cartridge The Filter Wheel Cartridge Dialog appears Filter Wheel Cartridge Dialog Name Cartridge Excitation 0 Cartridge Type Excitation C Emission Position Wavelength Bandwidth 45 z0 S30 ed 530 z 360 Define the characteristics for this filter wheel cartridge
96. an be very weak very strong or anywhere in between The photomultiplier PMT is the device inside a fluorometer that measures the light emitted from the sample For each assay the sensitivity gain of the PMT should be adjusted to ensure that the signals from all wells fall within the appropriate dynamic range of 0 to 99 999 relative fluorescence or luminescence units RFU or RLU The drawing on the left compares results with an optimum sensitivity setting with settings that are too low or too high Tips for selecting the optimum sensitivity gt Assays using 96 or fewer well plates typically require sensitivities between 75 and 190 Assays using 384 well or other higher density plates will likely require higher sensitivities because of the narrower optical probes gt One method for determining the optimum sensitivity for an assay is to define multiple filter sets with the same parameters varying only the sensitivity setting Another method is to enable KCjunior s Automatic Sensitivity Adjustment feature see Specifying Filter Options gt As the sensitivity setting increases the measurement values increase If many wells result in OVERFLOW indicating RFU or RLU values greater than 99 999 the sensitivity setting is probably too high gt If each well contains more than one fluorophore one of which might give brighter or weaker results assign each filter set its own sensitivity gt Ifthe Sensitivity
97. and displays the results Sample test results are shown on the next page 9 8 e Utilities User s Guide to KCjunior System Test Results Fa 2647 2350 2593 2662 2534 2327 550 Gain 17421 53702 38297 35379 44428 39411 38769 1180 1356 L207 1328 1363 1299 L195 z 750 Gain 16602 54648 39177 36356 45400 40591 39897 636 724 649 710 728 695 644 Noise Max 4878 5629 4990 5511 5662 5387 4942 Noise Min 4877 5627 4988 5510 5661 5386 4941 02 05AM 11 05 99 INCUBATOR SELF TEST Temperature Setpoint 0 0 Current Average A D Test T Z673 Min at ade Range Thermistor 2e enl Min Sal a ey Range Thermistor Sieceire 6 Min 6 eae ag eae Range Thermistor 4 27 2 Min 2 Se aS LR Range Thermistor SYSTEM TEST PASS gt Results should show SYSTEM TEST PASS gt If the test fails and the reader begins beeping press the Stop button on the reader The reader should then send an error code to KCjunior resembling the following SYSTEM TEST FAIL ERROR 0302 DETECTED Refer to the reader s user manual for a list of error codes and their potential causes gt Click Print to print the results or OK to close the results Test results are automatically saved in the Results Database Reader System Test History When a Reader System Test is complete the test results are automatically saved to the Results Database These test results can viewed at any time To view historical Reader System Test results 1 Sele
98. ange RFU or RLU 2 Specify the High Value This is the fluorescence or luminescence value that the High Wells should approach but not exceed gt The default setting is 90 000 RFU RLU and the maximum value is 99 999 Practically this value could be anywhere from 10 000 to 90 000 3 If this is Filter Set 1 checking Use Result for All Filter Sets instructs KCjunior to use the sensitivity determined here for all other filter sets with Automatic Sensitivity Adjustment enabled Ejecting the Plate Between Filter Sets For fluorescence luminescence protocols enable Eject between filter set reads to e Provide the ability to add reagent to the same plate between readings e Enable the reading of an initial blank plate followed by the sample plate e Enable the reading of up to 6 different plates using one protocol 5 30 e Defining Protocols User s Guide to KCjunior Let KCyunior automatically calculate the minimum Kinetic Interval Note If shaking is enabled the Shake Duration is included in the calculation of the Total Read Time If the reader is set to run in Rapid Mode the minimum kinetic interval can be set to a lower value than the theoretical minimum value calculated by KCjunior Refer to the reader s user manual for instructions on changing the reading mode The reader does not need to be connected to the computer to select a Plate Geometry User s Guide to KCjunior Defining Kinetic Parameters
99. ansformations Defining 5 46 Deleting stored 9 22 Formula syntax 5 50 Plate formula 5 46 5 49 Transformation formula 5 46 5 47 Tutorial 2 15 3 12 U Under indication 7 37 Uninstalling KCjunior 2 3 Universal plate test 9 10 Universal plate test history 9 14 Use result for all filter sets 5 30 Utilities Carrier Out In 9 4 Database maintenance 9 14 Diagnostics 9 8 9 13 Dispenser control panel 9 4 9 7 Incubation control 9 1 9 2 Reader system test 9 8 Universal plate test 9 10 Utilities menu 3 5 Index e 13 7 Vv Validate a formula 5 48 5 49 Validate read method parameters 5 31 View menu 3 7 W Wavelength function WV 5 52 7 19 7 20 T21 129 Wavelengths Multiple 5 16 Primary 5 16 Reference 5 16 Retrieving from reader 5 16 5 36 Well identifiers 4 5 5 38 Well IDs Assigning 5 38 5 39 Defining defaults 5 39 Modifying an existing template 5 43 Referencing in a formula 5 53 Template 5 38 Well information 7 36 Well location referencing in a formula 5 52 Well mode reading 5 21 Well notations 7 1 7 2 Well reference 5 52 Well type colors 4 5 4 9 Windows operating system 2 2 WV wavelength function 5 52 7 19 7 20 L21 1 29 X X in a formula 5 53 13 8 e Index User s Guide to KCjunior
100. arameter curve methods however the logarithmic scale is applied only to the plotting of the curve The curve is calculated using linear numbers Specify an X Axis Label using up to 32 alphanumeric characters This label appears on displayed and printed reports gt Acommon label is Concentration If the Standards are defined with multiple replicates enable standard averaging by clicking Average Standards gt If Average Standards is enabled KCjunior uses the average of the standard replicates when generating the curve gt If Average Standards is disabled KCjunior uses the individual standard replicates when generating the curve gt Note The Point to Point and Cubic Spline curve methods automatically use the average of the standard replicates If required click Extrapolate to display extrapolated concentrations unknown values on the Y axis which are outside the range of the standards gt On data displays and reports extrapolated concentrations are enclosed by parenthesis for example 27 345 gt Note The Cubic Spline curve method does not permit the display of extrapolated concentrations Optional To calculate the concentration for a specific value enable Calculate Interpolation Value then enter the desired value Valid Interpolation Values include 0 and 1 0000e 99 to 9 9999e 99 gt Ifa curve generates successfully KCjunior calculates a concentration for the entered Interpolation Value T
101. arks are the property of their respective holders Restrictions and Liabilities Limited Software Warranty Information in this document is subject to change and does not represent a commitment by Bio Tek Instruments Inc Changes made to the information in this document will be incorporated in new editions of the publication No responsibility is assumed by Bio Tek for the use or reliability of software or equipment that is not supplied by Bio Tek or its affiliated dealers Bio Tek Instruments Inc Bio Tek warrants for a period of ninety 90 days after the date of delivery that all software manufactured and sold by Bio Tek the Software will conform as to all substantial operational features to Bio Tek s then current published specifications meaning those specifications in effect as of the date of purchase and will be free of defects which in Bio Tek s sole judgment substantially affect system performance This Warranty is limited to the original purchaser the Purchaser and cannot be assigned or transferred All claims under this Limited Warranty must be made in writing or fax to Bio Tek attention Service Department The Purchaser must notify Bio Tek in writing within ninety 90 days after the date of delivery of the Software to the Purchaser of any claim under this Limited Warranty This 90 day period shall not be extended by the delivery or receipt of any subsequent modifications to the Software
102. asily between them Retrieve optical density fluorescence or luminescence data from a wide variety of Bio Tek s microplate readers Perform Endpoint Kinetic MultitWavelength and Spectral Scan reads as well as Dispense Read sequences MultiWavelength Spectral Scan and Dispense Read capabilities are reader dependent Perform Fluorescence and Luminescence reads with Bio Tek s FLx800 Create protocols that contain instructions for the operation of the attached microplate reader and for analyzing data retrieved from the reader Display print export save and archive results Export plate data to a file or directly to a Microsoft Excel spreadsheet Share protocol and results databases among multiple KCjunior users Define two levels of formulas to manipulate raw or blanked data Collect data from Bio Tek s automated microplate readers in the standard 8x12 format as well as in 2x3 3x4 4x6 6x8 6x10 T 6x12 T 8x12 T 8x12 H 8x12 M and 16x24 formats reader dependent Correct the wells on a microplate by a constant value to effectively eliminate background absorbance Introduction e 1 1 1 2 e Introduction Normalize absorbances to 1 cm by using the Pathlength Correction feature Calculate the mean standard deviation and coefficient of variation of any group of wells on the plate Generate a linear quadratic cubic point to point 2P 4P or cubic spline standard curve Calculate concen
103. aster Protocol Database mdb Browse Cancel Help In the Protocol Database Pathname field enter the pathname drive directory and file name of the database containing the protocols to be imported Alternatively use the Browse feature to locate and select the database Click OK The Import Protocols dialog appears listing all protocols in the database User s Guide to KCjunior Import Protocols Dialog Protocal Hame Import Protocal s k Cancel Help 340 Gluco Cell Count Color Comb B 0 5 Use the mouse to select the protocol s to be imported gt Press the Shift key while using the mouse to select contiguous protocols gt Press the Ctrl key while using the mouse to select non contiguous protocols 6 Click the Import Protocol s button The protocol import begins gt Ifa duplicate Protocol Name is detected KCjunior does not overwrite the protocol User s Guide to KCjunior Defining Protocols e 5 7 Defining Protocol Parameters 5 8 e Defining Protocols When creating a new protocol a minimum set of General Information and Read Method parameters must be defined for the protocol to be valid In addition Templates Curve parameters Cutoff criteria and Report options can be specified Defining Protocol Parameters describes every step of the protocol definition process Defining General Information General Information includes protocol identification minimum maximum limit s
104. asure The default is mOD min 5 Click OK The kinetic results redisplay The Maximum Slope automatically recalculates based on the new Points Slope value Raw and Blanked Spectral Data Raw and blanked spectral scan data are viewed under the Raw Data tab View options include sets of ODs at each wavelength the spectral curve for each well and the peak OD and corresponding wavelength for each well To switch between the different views click the appropriate Scan View Options radio button Scan view Options f Raw Data C Spectral Chart C Blanked Data C Peak ODA avelength Individual Spectral Reads Raw Data results for individual spectral reads are shown below Raw Data Template Wavelength Scan View Options z Calculation Options 440 Raw Data Spectral Chart Spectral Chart Blanked Diss C Peak OD Wavelength __Specttal Chart __ Spectral data is initially displayed in this format reporting the Raw OD read for each well at the First Scan Wavelength e To view the Raw ODs for a different wavelength enter the desired wavelength value in the Wavelength field or use the up down arrow buttons e Ifa blank well was defined in the Well ID template click the Blanked Data radio button to view the ODs after blank subtraction Interpreting Results e 7 9 e Click the Calculation Options button to adjust the wavelength range used for calculations for all scanned wells
105. avelength range on each of up to 8 wells A graph showing absorbance or transmission vs wavelength is reported for each well To define the Spectral Scan wavelength range 1 Select the Spectral Scan Read Method Type 2 Enter the First Scan Wavelength and Last Scan Wavelength The valid ranges for the First and Last Scan Wavelength fields are 200 998 nm and 201 999 nm respectively The First setting must be lower than the Last setting 3 Set the Scan Increment to determine which wavelengths will be used for measurement within the wavelength range For example if the wavelength range is 200 600 nm with a Scan Increment of 10 nm measurements are taken at 200 210 220 600 nm Defining the Number of Plates to be Read Up to 100 successive plate reads can be performed with one Endpoint protocol The plate carrier is automatically ejected after each read to allow for submittal of a different plate To define the Number of Plates to be read 1 Enter the number of plates to be read in the Number of Plates field gt Atrun time KCjunior provides the option to assign a Plate ID to each individual plate gt If the attached reader is equipped with a plate barcode scanner and Use reader s barcode scanner for plate ID is selected under SetupIConfiguration General KCjunior scans the plate before each read to capture the plate ID gt The raw data for all of the endpoint reads are stored as one record under one Result
106. btaining a password if you do not have Internet access Call fax or email Bio Tek s Customer Service department Tell them you wish to obtain a password for KCjunior and give them your System ID 888 451 5171 To call from anywhere within the United States except from Vermont 802 655 4040 To call from outside the United States or from within Vermont 802 655 7941 To fax from anywhere sales biotek com Email address Registering the software if you do not have Internet access Be sure to register your copy of KCjunior with Bio Tek to record your warranty and to receive information on upgrades and new products If you cannot register online you can do so by filling out and mailing the postage paid registration card attached to the Customer Assurance Plan included in your software package Installing the Software e 2 17 2 18 e Installing the Software User s Guide to KCjunior Getting Started KCjunior s Menu Structure KCyjunior s functions are controlled through the main screen shown below User s Guide to KCjunior Getting Started e 3 1 3 2 e Getting Started Toolbar Use the icons on the left side of KCjunior s toolbar when working with results records alae 340 Glucose Aflatox AT Il B12 Assay Cell Count Cell Growth Color e A results record contains the raw data retrieved from the microplate reader and related information such as Results ID Plate ID plate read time s
107. col 2 Click the Template tab The Well ID Template appears 3 Define or modify Well ID information or click the Dilutions Transformations or Sample IDs radio button to select a different template type After a template type is selected related definable parameters automatically become accessible For example if the Well ID Template is selected data entry fields such as Well Type Selection Blank Method and Replicate Number become accessible Regardless of the template type the information entry screens are conveniently arranged with rows and columns that align with the selected Plate Geometry A sample Template information entry screen for the 8x12 plate geometry is shown below EmA A ge a ea ee ge ed ee ee Se jelk SMP11 SMP35 SMP35 SMP12 SMP36 SMP36 SMP13 SMP37 SMP37 SMP14 SMP38 SMP38 SMP15 SMP39 SMP39 SMP16 SMP40 SMP40 SMP17 SMP41 SMP41 SMP18 SMP42 SMP42 Instructions for defining Default Well IDs and designing Templates are provided on the following pages 5 38 e Defining Protocols User s Guide to KCjunior Define the default Well IDs before designing Templates Note The parameters available within the Well ID Template for the Spectral Scan reading method are limited to well type selections of Blank and Sample and Sample Numbering One to eight wells can be scanned and the wells do not have to be contiguous Design t
108. col database may be moved to a network to be shared by multiple users and renamed as NTWRKPRT MDB Setting Up the System e 4 9 4 10 e Setting Up the System To reconfigure the Results or Protocol Database Pathname L 2 Close any open results records and protocols Select Setup Configuration Click the Pathnames tab The Pathnames dialog appears KCjunior Configuration Well IDs Reporting Options General Pathnames Filter Wheel Cartridges Help Results Database Path Ic KCjunior D atabase Riesults mdb Browse Protocol Database Path C KCjunior D atabase Protocol mdb Browse MV Allow pathname selection at program start j Cancel In the Results Database Path or Protocol Database Path field enter the new location and or name manually or use the Browse button to search through the directory structure to locate the desired database Optional KCjunior provides an option to present the Pathnames dialog each time the software is loaded allowing the user to change the database pathname s To enable this option check Allow Pathname Selection at Program Start Click another tab to configure additional system parameters or click OK to save the changes close the dialog and return to the main screen Database Version A small number of KCjunior features such as Spectral Scanning user enabled Scientific Notation and Pathlength Correction require the protocol and or results databas
109. ct Utilities Diagnostics Reader System Test History The View Test dialog appears listing the test results View Test Dialog Test Results View Test 11 5 99 2 54 26 PM 100413 17 5 99 2 58 32 PM 100413 17 5 99 3 05 25 PM 100414 1145499 3 1 men 100415 hep Ss gt The list is sorted by date To switch between ascending and descending sort order click the Test Results column header User s Guide to KCjunior Utilities e 9 9 9 10 e Utilities Highlight the desired test then click the View Test button to view the results Click OK to close the results and return to the View Test dialog View another test or click the Exit button to return to the main screen Universal Plate Test The Universal Plate Test utilizes Bio Tek s Universal Calibration Test Plate to validate and document the consistency of reader alignment accuracy reproducibility and linearity For most readers this test can be performed from within KCyjunior To perform the Universal Plate Test L Ensure that the reader is properly connected to the computer and that all communication parameters are set correctly The reader should be equipped with the necessary filters to perform the test Refer to the calibration data table that came with the test plate to determine the required wavelengths Select Utilities Diagnostics Universal Plate Test to access the Universal Test Plate Dialog Examine the contents of the Universal Plate
110. cted High Value see step 5 gt High Wells are usually the highest standards or high positive controls and are expected to yield a high but not overrange RFU or RLU gt This range must not overlap the Low Well Range It can however overlap the Test Well Range User s Guide to KCjunior Defining Protocols e 5 29 4 Specify the Base Value This is the fluorescence or luminescence value that the Low and Test Wells must exceed during sensitivity determination to ensure that the plate produces values that are large enough to be meaningful gt The default and recommended setting is 100 RFU RLU 5 Specify the High Value This is the fluorescence or luminescence value that the High Well s should approach but not exceed in order to produce a valid signal to noise ratio calculation gt The default setting is 90 000 RFU RLU and the maximum value is 99 999 Practically this value could be anywhere from 10 000 to 90 000 6 If this is Filter Set 1 checking Use Result for All Filter Sets instructs KCjunior to use the sensitivity determined here for all other filter sets with Automatic Sensitivity Adjustment enabled To enable Automatic Sensitivity Adjustment and define the pre reading parameters for the Scale to High Well option 1 Specify the High Well This is the target well to be measured gt The High Well usually represents a high standard or high positive control and is expected to yield a high but not overr
111. ctions for analyzing data retrieved from the reader Reading a Plate Without a Protocol If a fast read is required that is with no protocol open KCjunior requests minimal information then initiates the plate read Following the read a protocol can be created or opened to analyze the data Note The spectral scan read method requires a protocol to define the wells to be scanned To perform a Fast Read 1 Ensure that the reader is properly attached to the computer and that the correct reader type and communication parameters are defined under Setup Reader See Setting up the System in Getting Started for more information 2 Initiate the read by clicking the Read Plate button in the KCjunior main screen selecting Results Read Plate or by clicking the toolbar icon The Read Plate dialog appears Read Plate Dialog Reader 7 ja uant Simulation Data Protocol Mame Read From File Oligonucleotide Results 1D Help Oligonucleotide Flate Description LOT 765b Reading Plates e 6 7 3 Enter a Results ID using up to 32 alphanumeric characters The ID is optional in this dialog but it will be required later if the results are to be stored in the Results Database gt If performing a multiple plate endpoint read all of the plates in the read will be stored in the Results Database in one record under this Result ID 4 Optional Enter a Plate Description using up to 1024 a
112. ctive until terminated You may terminate it by destroying the programs and documentation and all copies thereof This license will also terminate if you fail to comply with any term or condition of this Agreement You agree upon such termination to destroy all copies of the programs and documentation Rev Date Changes A April 1998 First Issue B May 1999 Updated Defining General Information to include precision and scientific notation Updated System Requirements to include the Macintosh Enhanced requirements for operating system and display resolution Updated Defining Protocols Read Method Parameters to include pathlength correction and spectral scanning Updated Defining Protocols Reports to include information on the new Curve Report Updated Reading Plates to include collecting data from the EL301 reader Added Appendix A Sample Reports C December 1999 D January 2001 E October 2002 Added support throughout for fluorescence luminescence readers Updated Defining Protocols Reports to discuss enhanced Matrix Reports Added new sample reports to Appendix A Updated Installing the Software to introduce a new method for password distribution and software registration Updated Setting Up the System to introduce dual reader support and the option to pre configure Filter Wheel Cartridges for the FLx800 Updated Defining Protocols to introduce new features including partial plate reading
113. d Blank Data columns contain the raw and blanked values for the first defined filter set e If viewing statistics for a titer protocol click the Titer Results button to view curve parameters r square values and interpolation results for all samples on the plate e The information provided in the Statistics dialog depends on the protocol parameters The following may be displayed Well ID The identifier assigned to each well Loc The location of the Well ID in the plate Note If viewing statistics for a protocol that uses a stored curve the Loc values for the standards will contain User s Guide to KCjunior User s Guide to KCjunior Blank Data Calc Conc Corrected Concentration Dilution Label Max Slope Mean Raw Data Std Dev CV Transform Y Axis Data If the protocol specifies blanking and does not contain transformation formulas the Blank Data will be displayed If concentrations were calculated they will be displayed in the Cale Conc column If Pathlength Correction is enabled in the protocol MultiWavelength reads only to normalize absorbances to 1 cm the Corrected ODs will be displayed If viewing statistics for a protocol that uses a stored curve the concentration values as defined in the protocol will be displayed in the Concentration column If dilutions were assigned in the protocol they will be displayed in the Dilutions column If cutoff criteria are specified t
114. d well IDs such as STDO1 0 800 Multiple cutoffs must be entered in ascending order down the screen In the following example the NC mean must equate to a value that is less than that of the PC mean for the cutoff evaluation to be valid User s Guide to KCjunior User s Guide to KCjunior Cutoff Lowest Value INC p Fe Ea es Highest Value ss eal 4 Click the E button to the right of a Cutoff field to let KCjunior assist with formula definition and verification The Formula dialog appears Formula Dialog Formula STD01 0 800 Cancel Help Functions well IDs SD CY T Locations pa Ea JAN gt Assemble the formula using a combination of Functions Well IDs Mathematical values and symbols and Location indicators gt Functions and Well IDs can be entered manually or they can be selected from the list boxes A Function performs a specific operation on the contents inside the parenthesis such as the calculation of a mean value or the Standard Deviation The Well IDs list contains those Well IDs that are valid for this protocol Each Well ID represents the mean of its replicate group To add an item from a list box to the Formula field highlight the desired item then double click To insert an item between two existing items in the Formula field begin by clicking the mouse once in the Formula field where the new item should appear Highlight the desired item in the list box
115. dentifiers are To accommodate a variety of applications KCyunior provides the assigned to microplate wells flexibility to customize references to the different well types These within the Well ID customized references are called Well Identifiers Template See Designing a Well ID Template in e For example a well that KCjunior consistently treats as a Defining Protocols for Standard behind the scenes can be identified as STDO1 or more information STDRDO1 or CALO3 in a Well ID Template in results displays and on printed reports To configure Well Identifiers 1 Select Setup Configuration The Well IDs dialog appears KCjunior Configuration x Well IDs Reporting Options General Pathnames Filter Wheel Cartridges Help Selection Blank Blank Standard Standard Sample Sample Control 1 Conti Control 2 Con2 Control 3 Conti Control 4 Positive Control Control 5 Negative Control am ras a m Th m Cancel 2 Enter a Selection name and its associated Well ID for each default well identifier gt The Selection name is the descriptive link between the generic well type 1 e Blank Standard Sample Control and the 2 to 5 character Well ID Each Selection can contain up to 16 alphanumeric characters including spaces gt The Well ID is the short identifier that appears in results displays and on printed reports It can also be used in transf
116. ding This option typically applies to the kinetic read method User s Guide to KCjunior Not every reader that supports plate shaking supports the Variable intensity setting User s Guide to KCjunior 3 Select the Shake Intensity Available options include Low Medium High and Variable If Variable is selected the plate is shaken at varying intensities over the defined duration 4 Set the Shake Duration The valid range is 1 60 seconds Optional For kinetic protocols if Shake Every Read is enabled the Continuous Shake option is available If selected the plate shakes continuously between the kinetic reads Selecting a Read Mode If the currently selected reader supports multiple Read Modes choose a mode of Normal Rapid or Sweep e Normal mode is the default and its exact behavior is reader dependent In general this mode results in slower plate reading and yields the most precise results e With Rapid mode reading is faster than with Normal mode e With Sweep mode reading is very fast because the carrier sweeps the plate through the reader without stopping Notes The selection of one read mode over another generally depends on the desired outcome of the assay better precision versus faster plate reading All readers that support multiple read modes support the Normal mode Many readers also support the Rapid mode The PowerWave Robotic instrument supports the Sweep mode more readers may support
117. displayed in this format reporting the raw values for each well of Kinetic Read 1 e For single wavelength absorbance reads each well contains the OD measured at the Primary wavelength e For dual wavelength absorbance reads each well contains the delta OD value calculated by the reader The reader calculates the delta OD by subtracting the measurement at the Reference wavelength from the measurement at the Primary wavelength e For fluorescence or luminescence reads each well contains the RFU or RLU value calculated for the the first filter set defined in the protocol If two filter sets are defined use the Filter Set drop down menu to choose a different filter set Interpreting Results e 7 5 e If blank wells were defined in the Well ID template click the Blanked Data radio button to view the data after blank subtraction e To view the results for a different kinetic read enter the desired read number in the Kinetic Read field or click the up down arrow buttons e Click the Calculation Options button to adjust the kinetic read limits to alter the maximum slope calculation or to choose a different maximum slope unit of measure Kinetic Curves Kinetic read results are shown below in the Kinetic Curve format To print the Kinetic Curves Raw Data Template display as shown here Press Alt Print Screen to copy the screen shot to the Clipboard paste the Clipboard contents into for example an empty word proce
118. e Plate Letter Data for File 4 Plate A Strips 1 to 6 will be sent to KCjunior Reading Plates e 6 15 gt Do not position the cursor under the File Number as shown below Fa W1 MF 1A A1 2 202 gt Consult the EL301 Operator s Manual for more information on Data Output to Peripheral Devices 7 Data retrieved from the EL301 can be analyzed manipulated saved and printed in the same manner as any other set of plate data in KCjunior gt Overrange reads unread strips and unread wells appear as eR in KCjunior Reader Runtime Errors If the reader experiences an error KCjunior attempts to perform data reduction with any data that it received prior to the failure For example if the reader experiences an error during the 10 of 12 kinetic reads results are generated based on reads 1 through 9 KCjunior also displays appropriate error messages with the results e Refer to the reader s user guide for information on error messages displayed on the reader e See Communication Errors in the Troubleshooting section for more information on KCjunior s error and warning messages Comments Warnings Comments Warnings Following data reduction if an error is detected or a condition is present that requires notification a Comments Warnings button becomes available in the results display Click this button to see the comments and or warnings generated for the plate e See Comments and Warnings in the Troub
119. e Delete Protocols s 340 Gluco Aflatox Cancel T III B12 Assay He Cell Count Select the results record s or protocol s to be deleted gt If deleting results records click the Results and Creation Date column headers to sort the records alphabetically or by date gt Use the mouse to select the item s to be deleted Press the Shift key while using the mouse to select contiguous items Press the Ctrl key while using the mouse to select non contiguous items Click the Delete Results or Delete Protocol s button The selected items are permanently removed from the current database Utilities e 9 19 9 20 e Utilities Deleting Stored Curves After stored curves have been safely archived or if certain curves are no longer needed they can be deleted from the current Results Database To delete stored curves L 2 Close any open protocols Select Utilities Database Maintenance Delete Stored Curves The Delete Stored Curves dialog appears listing all curves stored in the current Results Database Delete Stored Curves Dialog Ea Delete Curvefs 4P Curve 1 4P Curve 2 Cancel Linear Curve 1 Help Quad 1 Select the curve s to be deleted gt Use the mouse to select the items s to be deleted Press the Shift key while using the mouse to select contiguous curves Press the Ctrl key while using the mouse to select non contiguous curves Click the Delete Curve s button The selec
120. e X BLK subtracts the BLK mean from the current well The operator represents multiplication e SMP01 10 multiplies the mean of the sample replicate group SMPO by 10 e X 100 multiplies the current well by 100 The operator represents division e PC NC divides the PC mean by the NC mean e X A2 divides the current well by the value in well A2 Parentheses represent an inclusive unit for calculation e PC NC 100 divides the difference between the PC mean and the NC mean by 100 e B4 STD1 100 divides well B4 by the STD1 mean then multiplies the result by 100 The CV function calculates the coefficient of variation of the contents inside the parenthesis The CV represents the standard deviation as a percentage of the mean standard deviation mean 100 The CV function supports up to five parameters each of which must equate to a single value e CV 3 124 3 094 2 997 returns 2 161 e CV CTRL1 CTRL2 returns the coefficient of variation for all replicates of both control groups If there is a single parameter inside the parenthesis then it is assumed to be a Well ID e CV PC returns the coefficient of variation for the group PC User s Guide to KCjunior DIL FS MEAN User s Guide to KCjunior The DIL function brings forth the currently assigned dilution value in the well This value can then be included in a formula The DIL function has no parameters associated with it It returns a
121. e program files e 1 MB for every set of 250 plates 96 well geometry stored on the hard drive e 3 1 2 high density diskette drive 1 44 MB or CD ROM drive e 16 color display 640x480 800x600 recommended e Keyboard mouse and serial ports Important e KCyjunior is capable of running any protocol on a computer meeting the minimum requirements if KCjunior is the only executing software If additional software including user programs browsers network connections scanning software and or utilities is processing concurrently with KCjunior overall performance may be affected 2 2 e Installing the Software User s Guide to KCjunior Removing an Earlier Version of KCjunior If you have an earlier version of KCjunior installed on your computer you should remove it before installing the new version For optimal performance only one version of KCjunior should reside on your computer at any one time Note This process does NOT remove existing protocol or results databases from your computer To remove KCjunior For optimal performance 1 Start Windows only one version of A KCjunior should reside on your computer at any one time Note If you are prompted with the Windows password entry enter a valid password If you are not prompted with the Windows password entry disregard this note 2 Click the Windows Start button then select Settings Control Panel I Control Panel a Printers e A Taskbar
122. e scanner enable Use Reader s Barcode Scanner for Plate ID to scan the barcode label on the plate before it is read gt If this option is enabled KCjunior attempts to scan the barcode when the plate is pulled into the reader If the read is unsuccessful KCjunior then asks the operator to enter the Plate ID manually gt The scanned or manually entered Plate ID is available as a part of the report export header information gt This option is supported for single and multi plate reads It is not supported for the empty plate s of a Multi Wavelength read gt Ifthe barcode returned from the reader is preceded by an unnecessary identification character you can check Remove first character of barcode to have KCjunior remove it 5 Click another tab to configure additional system parameters or click OK to save the change close the dialog and return to the KCjunior main screen Pathnames Results records and protocols are stored in designated Results and Protocol Databases respectively When opening or closing results records and protocols KCjunior automatically refers to the Results and Protocol Database Pathnames for retrieval and storage e Select Setup Configuration Pathnames to see the current configurations The default results and protocol databases are named RESULTS MDB and PROTOCOL MDB respectively These databases can be copied moved or renamed using any file management software e For example the proto
123. e to be at or above a certain version number This is only a concern if databases were created with an earlier version of KCjunior A software upgrade may contain new features that require newer databases The User s Guide and Help System show the minimum required database version number next to each feature that has such a requirement User s Guide to KCjunior e To see the version numbers of the currently configured protocol and results databases select Help About KCjunior About KCjunior Ea K C KCjunior Installation v1 20 BTILab32 dll v1 20 Reader Control Version KCJriwin exe v1 20 Results Database Version v1 3 Protocol Database Version 1 3 This product is licensed to BTE3135 Bio Tek Instruments Inc 13135 Copyright Bio Tek Instruments Inc 1998 1999 All Rights Reserved gt Protocols and results records from an older database can be imported into a newer database with a higher version number See Importing Protocols in Defining Protocols for more information Filter Wheel Cartridges If one or both of the defined readers is the FLx800 a fluorescence luminescence reader Filter Wheel Cartridges is available within the Setup Configuration set of tabs The FLx800 has one slot for a removable Excitation filter wheel cartridge and another for a removable Emission filter wheel cartridge Each cartridge has four locations for filters or plugs A location can also be left open
124. ecessary microstrip reads 2 Connect the reader to the computer on which KCjunior is loaded gt Consult the EL301 Operator s Manual for instructions on Connecting the Model EL301 to an External Computer 6 14 e Reading Plates User s Guide to KCjunior User s Guide to KCjunior 3 In KCjunior define the Reader Setup parameters as follows gt Reader Type EL301 Com Port Communication port on computer Baud Rate 1200 Data Bits 8 Parity None Stop Bits 2 EOT Character 4 Open an existing or define a new protocol The Well ID Template should correspond with the type of strip s read 8 well strips Al to H1 A2 to H2 A12 to H12 12 well strips Al to A12 B1 to B12 Fl to F12 16 well strips Al to H2 A3 to H4 All to H12 Select Results Read Plate or click the Read Plate button KCjunior displays the prompt Press DATA OUT key on reader when ready Within the reader s main menu select the data to be transferred then press the DATA OUT key The optical density values are transferred to KCjunior gt On the EL301 display make sure the cursor is positioned under the Strip Number to send data for one strip or under the Plate Letter to send data for up to six strips F1 Wi MF 3A A1 2 202 The picture above shows the cursor positioned under the Strip Number Data for File 1 Plate A Strip 3 will be sent to KCjunior The picture above shows the cursor positioned under th
125. ecified the plate is read twice once at each wavelength During data reduction KCjunior automatically calculates the Delta OD as the difference between the two readings To select Primary and Reference Wavelengths Select a Read Method Type of Endpoint or Kinetic 2 Enter the Primary and Reference Wavelengths in the appropriate fields Alternatively click the Get Reader Wavelengths button to retrieve wavelength information from the reader The reader must be connected to the computer and communication parameters must be set properly Following successful retrieval use the drop down menus to select the Primary and Reference Wavelengths Selecting Multiple Wavelengths For certain reader models KCjunior provides the ability to read a plate at multiple up to six wavelengths Common reasons for reading a plate at multiple wavelengths include e Calculating the ratio of measurements at two different wavelengths e Determining a solution s peak absorbance by measuring at several wavelengths To select Multiple Wavelengths 1 Select a Read Method Type of MultiWavelength 2 Enter up to six wavelength values in the Wavelengths fields Alternatively click the Get Reader Wavelengths button to retrieve wavelength information from the reader The reader must be connected to the computer and communication parameters must be set properly Following successful retrieval click the drop down arrows to select the desired wave
126. ectly to Microsoft Excel to export the file to an Excel spreadsheet The Start Excel Now button and First Excel Cell Location option are presented See Exporting Data Directly to Microsoft Excel on page 6 21 User s Guide to KCjunior Select Results Export Data to open the Export Data dialog User s Guide to KCjunior Exporting Data to a File To export data to a file I From within the Export Data dialog choose the Export Destination option File Select the Data Option s to be exported gt Inthe Data Options box highlight the item s to be exported then click the Add button The selected item s will appear in the Selections box gt To remove an item from the Selections box highlight the item and click the Remove button or double click gt To insert a data option between two items in the Selection box highlight the item in the Selection box above which the new item will be inserted highlight the item in the Data Options box then click Add Check Output report header to include the Plate ID and the date and time of the plate reading Check Output data descriptions to include identifying information for each set of plate data gt See Sample Output with Report Header and Data Descriptions on page 6 23 Check Output empty row between plates to add a blank line after each set of plate data gt See Sample Output with Empty Row Between Plates on page 6 23 Enter a valid path and f
127. ed to assign the ID Now the template looks like this Ai 2 3 4a 5 je fec ue smon ismo ee t gt This is currently an invalid template To make the template valid two options are available Defining Protocols e 5 43 A dilution value of I indicates the sample is not diluted 1 1 A dilution value of 2 indicates the sample is diluted 1 2 The resulting concentration for the sample will be multiplied by 2 0 to achieve the true concentration 5 44 e Defining Protocols Option 1 With the Well Type Selection still set to Blank click wells Al and B1 to change them to BLANK The template is now valid Note If any transformations in this protocol referred to BLK all references would have to be changed to BLANK to ensure validity Option 2 Via Setup Configuration change the default Well ID for Blank wells back to BLK Return to the Well ID Template With Well Type Selection set to Blank click on well C1 to assign the ID BLK The template is now valid Defining Dilutions KCjunior provides the ability to assign dilutions to sample and control wells e In quantitative assays calculated concentrations are multiplied by the dilutions to produce the final concentrations e In titer assays for each sample group containing multiple dilutions KCjunior automatically attempts to generate a curve using the dilutions as concentrations To define Dilutions 1 Ensure that Sample and or
128. ed wavelength For each Location C1 D4 E2 F5 G3 H6 enter the OD associated with that well at the selected wavelength For readers that support spectral scanning a Peak Wavelength value is required Enter the value in nanometers nm Click OK to save the data sheet and return to the Universal Test Plate dialog Repeat the above steps to create a data sheet for each Universal Test Plate ID Wavelength to be tested User s Guide to KCjunior Universal Plate Test History When a Universal Plate Test is complete the test results are automatically saved in the Results Database These test results can viewed at any time To view historical Universal Plate Test results 1 Select Utilities Diagnostics Universal Plate Test History The View Test dialog appears listing all test results View Test Dialog Test Results i View Test 11 5 99 2 54 26 PM 100413 11 5 99 2 59 32 PM 100413 11 5 99 3 05 25 PM 100414 1175799 3 1 as 100415 _ Hep elp gt The list is sorted by date To switch between ascending and descending order click the Test Results column header 2 Highlight the desired test then click the View Test button to view the results Click OK to close the results and return to the View Test dialog 4 View another test or click the Exit button to return to the main screen User s Guide to KCjunior Utilities e 9 13 Database Maintenance Protocols results records stored curves system t
129. ees 7 19 CONCE NTA ONE 25s isi csn Sa cade T T E 7 20 The Standard CUYO anaipa aa AEA E AA 7 21 TEE UVE a a a a A E A meen com ecaxeaums 7 22 Triterac timo with tae Grr ap Misc ioc caces te a r S 7 24 Printings and Cop yine ne Graphsecssmrorgnn a a 7 25 Editing the Standard ur yess asec sts i E estates 7 25 Stornes the standard C NVG sasea a e Batman Geese 7 26 LADE IS E cas cse sara yeaa aes aaa sea ha csasa se a iatae tania ae aeateaiten 7 27 MG WMAP AGS o a hat Cer lath E Sats es tiated Cece heies aici aa tea eneed TA 7 28 Plate Formula Resulte ssrin anr E E AEA EAE S AEEA 7 29 SASU CE a ere 7 30 Auto SEMSIEIVILY RESUS ansia cnn Gidesaassl Meiaseules acenachannGddusaatddenaas 7 32 Automatic Sensitivity Adjustment Process ccccccccccssssssssssseeeeecceeeeeeeeeeaeaaeeeenees 7 33 Resu Jo 0G t ueeeereee meeyreenre a cer cree a a eter tian ete omerc ter a 7 34 Plate mormi Oese a aphdorecstalavtaddl ang aaah enh eeas toes 7 34 Well Infor iia tons 55 itasstousen st diet A tae elena ans enact leva 7 36 IN ais Kat WCU Sa daca cen cat ica Sea caneatcasauesGcamkcan A A 7 37 Modifying Simulation Data s s1ccsco0tciccdascoccssttjadasnavnacssseheceddarsaussaaioetassahecsiatieresian s 7 38 User s Guide to KCjunior Contents e iii Printing Reports TSC DOU e eu ateeisahat is alee a nan aee alates Utilities ONEIN IE rectal enccs ated ea EE AEA eet banat T A Incubation Controls reni a a Camie r Cisse ccleaner act eaonelecna
130. elength subtraction value of 0 600 OD falls outside the valid range Protocol database versions 1 2 and greater support the Precision parameters Select About KCjunior from the Help menu to see the version of the currently selected Protocol Database User s Guide to KCjunior Explanation During data reduction the OD RFU or RLU value in each well is verified against this range If the value falls outside the range two options are available gt Discard the out of range value If Discard Out of Range Values is selected the well is reported as OUT and is excluded from data reduction calculations For example if one replicate of a sample group is reported as OUT the sample group mean is based on the remaining in range replicates gt Retain the out of range value If Discard Out of Range Values is not selected KCjunior automatically sets the raw OD RFU or RLU value to the Minimum or Maximum Raw Data Limits value as appropriate The new value is displayed inside brackets 2 999 and is included in further calculations such as group mean calculation transformations and standard curve generation Optional Adjust the Blank Limits Minimum and Maximum Values to define an inclusive data range for blank wells The valid limits are reader dependent Explanation During data reduction the OD RFU or RLU value in each blank well is verified against this range If a value falls outside the range two options are avai
131. ells is subtracted from every well on the plate The average of the blank wells in the first half of the plate column oriented is subtracted from each well of the first half of the plate The average of the blank wells in the second half of the plate is subtracted from each well of the second half of the plate The average of the blank wells in the first quadrant of the plate column oriented is subtracted from each well of the first quadrant The average of the blank wells in the second quadrant is subtracted from each well of the second quadrant etc The average of the blank wells in each row is subtracted from each well of that row The average of the blank wells in each column is subtracted from each well of that column Each well of the plate is reduced by the value of a Blank Constant 9 If the template contains Standard wells define the associated Concentrations e Before defining concentrations ensure that the Well ID template contains Standard well IDs as required to support the desired curve fit type For more information see Defining Curve Parameters Selecting a Curve Method in Defining Protocols To define Concentrations click the Concentrations button from within the Well ID Template User s Guide to KCjunior Defining Protocols e 5 41 5 42 e Defining Protocols The Concentrations dialog appears Concentrations Dialog x STANDARD Conc Gy EA Cancel Help Startin
132. elp Menu provides access to KCjunior s help system and to Bio Tek s internet site Help Topics Gl Li a Bio Tek onthe Web Bio Tek Home Page e Register KCjunior Get Password Help Topics Access KCjunior s help system Contents Index and Find Bio Tek onthe Goto Bio Tek s home page or register the software Web and get your password online About KCjunior Retrieve software version information This is useful if contacting Bio Tek for support User s Guide to KCjunior Getting Started e 3 7 Navigating the Help System Most KCjunior dialogs contain a Help button that when clicked opens an informational Help dialog In addition to this context sensitive help KCjunior provides access to the entire Help system From the main screen select Help Help Topics Help Topics KCjunior Help El Ei Contents Index Find Click a book and then click Open Or click another tab such as Indes Help System Contents Introduction Ca Getting Started Ce Sample Protocols amp Results Records Ce Setting Up the System Var Defining Protocals Overview Ca Working with Protocols Ne Detining Protocol Parameters Defining Protocol Parameters Co General Information a Head Method F Ce Templates gt Curves Co Cutotts Open Print Cancel any e Double click on a book 2 to see a topic s sub topics e Double click on a page LH to view the text for a topic e Highlight a topic then click Open to view the t
133. emperature Reporting Always report temperature information Report temperature if incubation requested Never report temperature information m Graph Options MV Display Curve Legend M Graph Interpolation Result if applicable i Cancel 2 Set the Displayed Decimal Places value Values such as optical density measurements transformation results and concentrations are reported with the number of decimal places defined in this field gt The range is from 0 to 3 decimal places Setting Up the System e 4 7 4 8 o Setting Up the System 3 Ifthe reader supports incubation select a Temperature Reporting option for printed reports gt Select Always report temperature information to report the reader temperature even if a plate is read with a protocol that does not specify incubation gt Select Report temperature if incubation requested to report the reader temperature only when a plate is read with a protocol that specifies incubation gt Select Never report temperature information to suppress all temperature information 4 Specify Graph Options gt Check Display Curve Legend to include a legend on the curve display and report gt Check Graph Interpolation Result to plot the interpolation value on the curve display and report 5 Click another tab to configure additional system parameters or click OK to save the change close the dialog and return to the KCjunior main screen Gene
134. empty row between plates to add an empty row after each set of plate data gt See Sample Output with Empty Row Between Plates on page 6 23 Specify the location ex Al C3 K15 of the first cell in the current Microsoft Excel spreadsheet gt This is where the first entry will be made in the spreadsheet If Output Report Header is enabled the header is stored in this location followed by a blank row The first data value such as the contents of well A1 is stored below the specified first cell Subsequent values then fill in to the right of and below the first data value See the sample Excel spreadsheet after data export below Plate 17394 Date Created 11 30 2000 10 37 16 AM Raw Data 0 000 1 425 1 992 2 028 Tip If performing multiple exports to the same spreadsheet use the First Excel Cell Location and Output empty row between plates features to append one data set to another with a row between each set Click the Export button gt Note If exporting data for a multi plate endpoint read the Export Endpoint Plates dialog appears Export Endpoint Plates Dialog Number of Plates Cancel First Plate to Export Last Plate to Export Specify the First and Last plates to export then click OK to continue Export format All selected data sets are exported for the first plate then for the second plate and so on through the last plate KCjunior launches Excel if it is not al
135. enabled in KCjunior under Setup Configuration General KCjunior attempts to scan the plate If no barcode is detected or if the attached reader is not equipped with a barcode scanner the Plate ID can be entered manually gt Click the Abort Read button to halt the read in progress Data reduction is based on any data retrieved up to the time the button was clicked gt When the plate read is complete KCjunior automatically retrieves the measurements from the reader performs data reduction and displays the results See Viewing Results or Interpreting Results for more information 11 To save the results e Select Results Save Results or press CTRL S KCjunior requests a Results ID if one was not defined before the plate read Enter the ID using up to 32 alphanumeric characters This is the name under which the record will be stored in the Results Database Click OK to save the results 12 To apply a protocol to the results leave the results record open and do one of the following gt Open an existing protocol If the read method parameters are compatible KCjunior automatically recalculates the results If the read method parameters are not compatible KCjunior provides further instructions gt Create a new protocol KCjunior automatically transfers the read method parameters defined for the fast read into the new protocol User s Guide to KCjunior Reading Plates e 6 9 Read Plate 6 10 e Reading Plates Reading
136. ence Unit RFU or Relative Luminescence Unit RLU is reported for each well for each kinetic read In addition a graph showing RFU RLU vs Time is generated for each well MultiWavelength Read During a MultiWavelength read every well on the plate is read once at each defined wavelength up to 6 The optical density value measured at each wavelength is reported for each well Optical Density OD The amount of light at a specific wavelength that is absorbed by the chromophore It is the reciprocal of transmission 1 7 Protocol A protocol is one of many records in a protocol database containing user defined information for performing a plate read and data reduction RFU RLU RFU stands for Relative Fluorescence Unit RLU stands for Relative Luminescence Unit In KCjunior if the current reader is set to a fluorescence luminescence reader such as the FLx800 the Protocol Read Method dialog contains a Chemistry parameter If the Chemistry is set to Fluorescence the plate measurement values are reported as RFU ex 87954 RFU If the Chemistry is set to Luminescence the plate measurement values are reported as RLU ex 57697 RLU User s Guide to KCjunior Glossary e 12 3 R Square Also referred to as the Coefficient of Determination R Square indicates the strength of a regression function curve fit If the value is 1 0 then the fit is very strong meaning that all of the points lie on the curve Standard Deviat
137. ences an invalid location references an invalid Well ID attempted a Standard Deviation on a well group with an insufficient sample population attempted a Coefficient of Variation on a well group with an insufficient sample population The well is in a blank sector that contains blanks but none of them are valid Cutoffs could not be evaluated for the well A concentration had no valid solutions on the generated curve The curve could not be generated Interpreting Results e 7 3 Raw Data Raw Data represents the original measurements that were either retrieved from the microplate reader entered manually or read from a file The format of the Raw Data display differs based on the defined Read Method Endpoint Kinetic MulttWavelength or Spectral Scan as described on the following pages Raw Endpoint Read Data Raw Data results for an endpoint read are shown below Raw Data Blanked Data Pathlength Correction Transformation Results Concentrations Labels Template Plate Formula Results Comments Warnings e For single wavelength absorbance reads each well contains the OD measured at the Primary wavelength e For dual wavelength absorbance reads each well contains the delta OD value calculated by the reader The reader calculates the delta OD by subtracting the measurement at the Reference wavelength from the measurement at the Primary wavelength e For fluorescence and
138. ensitivity Adjustment feature as an alternative to the more manual method of defining multiple filter sets with varying sensitivity settings If this feature is enabled KCjunior instructs the reader to perform several pre readings on the microplate In general these readings are performed on user specified wells using incremental sensitivity settings Each increase in sensitivity raises the RFU RLU values of all of the wells Certain wells are monitored to ensure that they and hence the assay do not overrange KCjunior reviews the results of the pre readings and selects the optimum sensitivity for plate reading KCjunior provides two options for Automatic Sensitivity Adjustment e Scale to Low High Range If this feature is enabled the instrument performs several pre readings of the specified Low Test and High Wells With each reading the sensitivity gain of the photomultiplier is increased The target wells are monitored to ensure that they and hence the assay do not exceed the defined High Value KCjunior reviews the results of the pre readings and selects the optimum sensitivity the one that gives the best signal to noise ratio when comparing one higher set of wells with another lower set of wells The entire microplate is then read using this sensitivity setting User s Guide to KCjunior e Scale to High Well If this feature is enabled the instrument performs several pre readings of the specified High We
139. er temperature will begin to decrease to room temperature To set a timer EL312I and EL340I readers only gt Manually set the temperature on the EL312I or EL3401 Refer to the reader s user manual for instructions gt In KCjunior s Incubation dialog enter the Set Point temperature optional for visual effect only gt Click the Start button to start the timer gt When the timer reaches 0 KCjunior will provide an indication to place the plate in the carrier If the reader type specified under Setup Reader supports computer controlled operation of the plate carrier or door the Carrier Out and Carrier In options are accessible in the Utilities Menu Select Carrier Out to open the door or to extend the plate carrier Select Carrier In to close the door or to retract the plate carrier Dispenser Control Panel For the FLx800 reader with the optional reagent dispense capability KCjunior provides a Dispenser Control Panel The panel displays the current dispenser status and provides access to dispenser specific functions such as prime and purge 9 4 e Utilities To access the Dispenser Control Panel L Ensure that the reader is properly connected to the computer and that all communication parameters are set correctly Ensure that FLx800 is selected as the Current Reader Select Utilities Dispenser Control Panel User s Guide to KCjunior Dispenser Control Panel Sampling Windows Supported 2 Number
140. ersal Plate Test Results The Delete System Test Results or Delete Universal Plate Test Results dialog appears listing all test results stored in the current Results Database Delete System Test Results x Test Results Delete Test s 11 5 99 2 54 26 PM 100413 11 5 99 2 58 32 PM 100413 Exit 11 5 99 3 05 25 PM 100414 11 5 99 3 10 47 PM 100415 _ He Select the test s to be deleted gt Use the mouse to select the items s to be deleted Press the Shift key while using the mouse to select contiguous tests Press the Ctrl key while using the mouse to select non contiguous tests Click the Delete Test s button The selected item s are permanently removed from the current Results Database User s Guide to KCjunior Troubleshooting Overview There are three categories of error and warning messages that may appear in KCjunior e Reader errors that occur during a plate read e Communication errors between the computer and the attached microplate reader e Data reduction comments and warnings This section describes every communication error and data reduction warning or comment that may occur while running KCjunior and in many cases provides suggestions for fixing the error User s Guide to KCjunior Troubleshooting e 10 1 Reader Errors If the reader experiences an error during a plate read KCjunior may present a message resembling the following KCjunior N The reader has returned the followi
141. es of transformations can be defined within one protocol e A Transformation Formula is performed only on the well s to which it is assigned e A Plate Formula is automatically applied to every well on the plate Here you will apply Transformation Formulas to subtract the Blanked OD value from each well in column 4 from its corresponding well in column 3 Note KCyjunior automatically calculates formulas based on the Blanked Data if Blank wells are defined in the Well ID Template If no Blank wells are defined formulas are based on the Raw Data 1 Inthe Transformation Formula field enter X A 4 X current well subtraction A4 contents of well A4 indicates a well reference retains the column number 4 when copying the formula 2 Highlight well A3 then click the Apply button The formula should appear under SMPO1 in well A3 3 With the cursor on A3 click the right mouse button An editing menu should appear 4 Select Copy from the editing menu User s Guide to KCjunior User s Guide to KCjunior 5 Click and hold the cursor use the left mouse button on well B3 Drag the cursor to well H3 and release Wells B3 to H3 should now be highlighted 6 Click the right mouse button to open the editing menu then click Paste Well B3 should contain X B 4 well C3 should contain X C 4 and so on to H3 X H 4 7 Click the Sample IDs radio button to continue Sample IDs By default KCjunior refers to
142. es the RFU RLU values of all of the wells If the Automatic Sensitivity Adjustment option Scale to High Low Range is selected KCjunior compares the results of the pre readings and selects the sensitivity that gives the best signal to noise ratio when comparing one higher set of wells with another lower set of wells The entire microplate is then read using this sensitivity setting A graph showing the results of these pre readings can be viewed from the results dialog by clicking the Auto Sensitivity button The Auto Sensitivity Results dialog appears Auto Sensitivity Results Dialog Auto Sensitivity Results Help Print Copy M Filter Set 1 T Filter Set 2 Filter Set 1 l Filter Set 3 Filter Set 2 Filter Set 3 o 2 5 T 1 150 Sensitivity M Show Legend Reading at sensitivity 40 e This graph can be viewed during or following the automatic sensitivity adjustment process e ach point represents the signal to noise ratio calculated at a particular sensitivity setting e To change the filter set s shown in the graph select or deselect the Filter Set checkboxes e This graph is not saved with the plate Click Print Copy to print the graph or to copy paste it into another application User s Guide to KCjunior User s Guide to KCjunior If the message There is no valid auto sensitivity data to display appears an error occurred during the auto sensitivity adjustment process See
143. est results and universal plate test results can be copied into archive databases that can then be stored as a part of normal backup procedures During the archive process e Protocols are copied to an Archive Protocol Database e Results records stored curves system test results and universal plate test results are copied to an Archive Results Database Archived or otherwise unnecessary protocols results records stored curves stored transformations system test results and universal plate test results can be deleted from the Protocol and Results Databases currently in use A Note on Backup Procedures It is strongly recommended that you backup safely store your protocol and results databases regularly Here are some suggestions e Routinely copy the complete protocol and results databases from your hard drive to the network or to a floppy diskette Note Check to ensure that network files are automatically backed up See Pathnames under Setting Up the System to find out where these databases are stored gt Enhance this suggestion by first copying certain protocols results records stored curves and or test results to archive protocol and results databases Then copy the archive databases to the network or to a floppy diskette Continue to review Database Maintenance for instructions e Instead of keeping your protocol and results databases on your hard drive store them on the network See Sharing P
144. etting and precision options To define this information for a protocol 1 Choose to create or modify a protocol Select the General Information tab The General Information dialog appears Protocol Definition General Information Read Method Template Curve Cutoffs Reports Protocol Name SAMPLE Aflatoxin Last Modified fi 1 12 99 2 40 06 PM Protocol Description Aflatoxin Al Assay in Wheat Germ m Min Max Limits Minimum Value Maximum Yalue Raw Data Limits OD 3 999 3 999 lV Discard Qut of Range Values Blank Limits OD 000 0 01 0 M Discard Out of R ange Values m Precision T Scientific Notation Raw Data Blanks Displayed Decimal Places 3 MV Scientific Notation Transforms Concentrations 2 If this is a new protocol enter a Protocol Name using up to 32 alphanumeric characters The protocol will be stored in the current protocol database under this name 3 Optional Enter a Protocol Description using up to 1024 alphanumeric characters gt Text entered here appears at the top of the Protocol Report 4 Optional Adjust the Raw Data Limits Minimum and Maximum Values to define an inclusive data range for all wells The valid limits are reader dependent User s Guide to KCjunior The Raw Data Limits OD Minimum Value can be set to a number less than zero to accommodate dual wavelength reads For example if the Minimum Value is set to 0 500 OD a post dual wav
145. ew set of plate data begin by clicking on the Read Plate button in the KCjunior main screen selecting Results Read Plate or by clicking the toolbar icon The Read Plate dialog appears providing three options for generating the plate For more information see e Reading a Plate on page 6 7 e Simulating a Plate Read on page 6 12 e Reading Plate Data from a File on page 6 13 Before Reading a New Plate A microplate can be read with or without a Protocol A protocol contains information for the operation of the microplate reader as well as instructions for analyzing data retrieved from the reader To read a plate without a protocol to perform a fast read simply click the Read Plate button from within the Read Plate dialog KCjunior requests minimal read method information then initiates the plate read If desired a protocol can be opened or defined after the data is retrieved from the reader To define or open a protocol before reading a plate refer to the Defining Protocols section of this User s Guide Reading Plates e 6 3 6 4 e Reading Plates Saving and Closing Results Records After generating or modifying a plate save the information by selecting Results Save Results or by clicking the toolbar icon Results records are stored in the current Results Database KCjunior either automatically saves the record under the Results ID assigned prior to microplate reading or requests the entry of a Results ID
146. exclude it from calculations place the cursor over the desired well click the right mouse button then select Mask Well e To access more detailed information about the plate place the cursor in the matrix click the right mouse button then select Plate Information e To access more detailed information for a particular well place the cursor over the desired well click the right mouse button then select Well Information e Ifa Comments Warnings button is available click it to see the data reduction comments and or warnings generated for the plate e To export data to a file or directly into Microsoft Excel select Results Export Data e To copy the data to another application highlight the desired wells then press CTRL C Switch to the destination application then press CTRL V or select a Paste option 6 24 e Reading Plates User s Guide to KCjunior Interpreting Results Overview User s Guide to KCjunior When plate reading is complete or after simulation data has been entered or data has been read from a file KCjunior displays the results as shown below i KCjunior Results Protocols Utilities Setup Curent Reader View Help SAMPLE Result File majaj SAMPLE Protocol x Hoei Raw Data Blanked Data Pathlength Correction Transformation Results Concentrations Labels Template Plate Formula Results Comments Warnings 1 261 1 091 0 914 0 539 0 277 0 167
147. ext for the topic e Click the Index tab to perform a keyword search e Click the Find tab to search the entire help system for a specific word or phrase e Click Print to print the text for a topic e Click Cancel to close the Help Topics dialog and return to KCyjunior 3 8 e Getting Started User s Guide to KCjunior Essential Information User s Guide to KCjunior KCjunior allows you to define instructions for acquiring microplate data and for reducing and analyzing that data Protocol Definition The minimum required instructions for plate data acquisition are the read method parameters These parameters can be combined with more detailed data reduction instructions within a protocol In addition to storing read method parameters a protocol stores templates curve fitting parameters cutoff criteria and reporting options While a fully defined protocol is useful for controlling a microplate reader and for performing data analysis it is not a requirement that a protocol be defined prior to plate reading KCjunior can perform fast reads using just the basic user defined read method parameters Plate Data Acquisition KCjunior provides three methods for acquiring plate data retrieve results from a microplate reader read data in from an existing file or manually enter simulation data Refer to the following sections under Reading a Plate starting on page 6 7 for more information e Reading a Plate Without a Protocol
148. f rows and columns in a microplate For example a Plate Geometry of 8x12 represents the standard 96 well plate 8x12 is the default for every reader type Depending on the current reader additional geometry selections can include 2x3 3x4 4x6 6x8 6x10 Terasaki 6x12 Terasaki 8x12 Hellma 8x12 Metric 8x12 Terasaki 16x24 and BioCell Defining Protocols e 5 31 To select a Plate Geometry 1 Ensure that under Setup Reader the desired Reader Type is currently selected 2 Use the drop down menu to view a list of available geometry options for the current reader type 3 Click once on the desired Plate Geometry type Optional If partial plate reading is supported by the current reader click the button to the right of the Geometry field to define the well s to be read gt Define a rectangle of wells to be read by entering the First Well and Last Well or by highlighting the desired wells in the grid For example to read columns 4 through 7 of a 6x8 plate set First Well to A4 and Last Well to F7 or highlight columns 4 5 6 and 7 in the grid gt To set the First and Last Well values to coordinate with the currently selected Geometry click Read Full Plate gt Click OK to return to the Read Method dialog Setting a Time Delay Before Reading Set a Delay Before Read to Use this feature to set a delay between the time the operator initiates the allow time for stabilization plate read and the time the
149. f the acceptable min max data range and Discard Out of Range Values is not selected Under if the Read Value falls below the lower limit of the acceptable min max data range and Discard Out of Range Values is not selected Error if the reader determined the OD RFU or RLU of the well to be significantly out of range gt Ifthe Read Status is Over or Under the Adjusted Value contains the Raw Data Limits or Blank Limits Maximum or Minimum Value as defined in the protocol gt Click OK to close the dialog and return to the results display Masking Wells Within any results display individual wells can be masked or temporarily excluded from a recalculation of the results For example a standard replicate with an atypical absorbance can be temporarily masked and excluded from the recalculation of the standard curve To Mask a well L Place the cursor over the desired well and click the right mouse button once The Results Options menu will appear Select Mask Well to mask the current well If the read is kinetic the well will be masked only in the current kinetic read Select Mask All Reads to mask the current well across all kinetic reads gt Inthe Raw Data results display the value in a masked well appears with a line running through it ex 4275 gt In other results displays the well will be empty To Unmask a well L Place the cursor over the desired well and click the right mouse button The Results O
150. feature does not apply to titer curves or stored curves There are two ways to edit the standard curve 1 From within the Standard Curve dialog click once on the standard point to be excluded The selected standard will dim and the curve will automatically attempt to recalculate gt Restore an edited standard by clicking on it once The curve will automatically recalculate 2 Click the Edit Stds button to open the Edit Standards Dialog This dialog lists each standard with its Y Axis Value Concentration as defined in the protocol and current Status User s Guide to KCjunior Interpreting Results e 7 25 Edit Standards Dialog 10 STOO aor Eas ance Help Suppressed iE E E Ei a E E m gt Ifthe Status field is empty the standard is currently included in the curve calculation Click the checkbox to suppress the standard gt Ifthe Status field contains Suppressed the standard is currently suppressed Click the checkbox to restore the standard gt Click OK to close the dialog and return to the Standard Curve dialog The curve will automatically recalculate Storing the Standard Curve The standard curve can be stored for use by another protocol This feature does not apply to titer curves To store a standard curve L 7 26 e Interpreting Results From within the Standard Curve dialog click the Store the Curve button The Stored Curve dialog will appear Stored Cur
151. formation Detailed Well Information is available for any well from within the Raw Data results display To access this information for a particular well Click the Raw Data results tab 2 Place the cursor over the desired well and click the right mouse button The Results Options menu will appear 3 Select Well Information The Well Information dialog will appear Well Information Dialog Well Location Read Number Read Value Read Status Adjusted Value The Well Location represents the well s location in the microplate in row by column format For a multiple plate Endpoint read the Read Number represents the plate for which the raw data value in this well was measured If the read is Kinetic the Kinetic Read represents the read during which the raw data value in this well was measured For a MultiWavelength read the Wavelength field shows the wavelength associated with the current Read Value and Empty Read or Result as appropriate for the data set The Read Value is the original unchanged Raw Data value Note A Read Value of 99 99 or 99 999 indicates a well error see Read Status Error on the next page User s Guide to KCjunior User s Guide to KCjunior gt The possible Read Status values are OK if the well was read without error Out if the Read Value falls outside the acceptable min max data range and Discard Out of Range Values is selected Over if the Read Value exceeds the upper limit o
152. g Concentration Delta MV Factor Within the Concentrations dialog the Standard IDs that were defined in the template appear in sequential order in the STANDARD column The concentrations associated with each Standard ID appear in the CONC column Individual concentrations can be entered manually in the CONC column or they can be assigned using the Auto Numbering feature Enter the Starting Concentration the concentration value to be assigned to the first standard Note Valid concentration values include 0 and 1 0000e 99 to 9 9999e 99 Choose the method by which to increase the concentration values for subsequent standards Delta to increase each standard exactly by a defined value or Factor to increase standards by a defined factor value Enter the Delta or Factor value as appropriate Click the mouse button at the top of the CONC column to assign the concentrations Click OK to save the information and return to the Well ID Template 10 When the Well ID Template is complete the Dilutions Transformations and Sample ID Templates can be designed gt gt Click the Dilutions radio button to assign Dilutions Click the Transformations radio button to define Transformation and Plate Formulas Click the Sample IDs radio button to assign sample identifiers User s Guide to KCjunior User s Guide to KCjunior Modifying an Existing Well ID Template Before modifying an existing Well ID Template co
153. g these cells and to ensure that the data exports to the correct cells well is the KCjunior term referring to a location in the microplate and cell is the Microsoft Excel term referring to a location in the spreadsheet The spreadsheet may contain formulas that reference other cells in the spreadsheet Be sure to define the export parameters so that the correct set of plate data is sent to the correct cells KCjunior exports data starting with well A1 in the specified First Excel Cell Location Switch back to KCjunior Select the Data Option s to be exported gt In the Data Options box highlight the item s to be exported then click the Add button The selected item s will appear in the Selections box To remove an item from the Selections box highlight the item and click the Remove button or double click To insert a data option between two items in the Selection box highlight the item in the Selection box above which the new item will be inserted highlight the item in the Data Options box then click Add Check Output report header to include the Plate ID and the date and time of the plate reading Check Output data descriptions to include identifying information for each set of plate data gt See Sample Output with Report Header and Data Descriptions on page 6 23 Reading Plates e 6 21 First Excel Cell Location E2 M Output report header 6 22 e Reading Plates Check Output
154. ge needs to be installed or replaced it must first be moved to its Maintenance Position To do this from KCjunior 1 Select Utilities Dispenser Control Panel 2 Click Syringe Setup 3 Click Move Syringe to Maintenance Position The syringe plunger will move to its furthest from home position The syringe can then be disconnected from the drive bracket and unscrewed from the valve see the FLx800 Operator s or service manual for further details User s Guide to KCjunior Utilities e 9 7 Diagnostics The Reader System Test and the Universal Plate Test should be performed routinely to validate and document the consistency of a reader s optical and electronic performance alignment accuracy reproducibility and linearity Reader System Test The Reader System Test validates and documents the consistency of reader optical and electronic performance For most readers this test can be performed from within KCjunior To perform a Reader System Test 1 Ensure that the reader is properly connected to the computer and that all communication parameters are set correctly 2 From the Utilities menu select Diagnostics then Reader System Test The System Test dialog appears System Test Dialog Operator Cancel Reader Serial Humber Help i 00413 Comments weekly maintenance 3 Enter the Reader Serial Number and an appropriate Comment if desired 4 Click the Run Test button KCjunior performs the test
155. he Interpolation Value is displayed on the Standard Curve dialog and on the Standard Curve report Defining Protocols e 5 57 See Data Reduction and Analysis under Essential Information to review the calculation structure which determines the basis for cutoff evaluation 5 58 e Defining Protocols Cutoffs Cutoffs are used to classify results During data reduction KCjunior evaluates results against user defined cutoff criteria and assigns each well a user defined Label The basis for cutoff evaluation may be Raw Data Blanked Data data normalized by Pathlength Correction or Maximum Slope depending on the protocol definition See Essential Information in Getting Started to review the results calculation structure To define Cutoffs and Labels 1 Ifa cutoff value or formula contains references to specific Well IDs ensure that the IDs are defined in the Well ID Template 2 Access the Cutoffs dialog Protocol Definition General Information Read Method Template Curve Cutoffs Reports Cutoff Lowest Value sTo 07 al Highest Yalue Eoo o y g EA i Cancel 3 Enter the cutoff values or formulas in the Cutoff fields These are the criteria against which the plate data will be evaluated gt Up to four cutoffs can be specified Each cutoff can be any one of the following A simple numeric value such as 1 500 or 50000 A well ID such as NC or LPC A formula combining numeric values an
156. he Label will be displayed for every replicate and group mean The Max Slope is reported for kinetic protocols The Mean is automatically calculated for every multiple replicate standard control blank and sample group Note The Mean is not displayed for samples controls with multiple dilutions If the protocol does not specify blanking or contain transformation formulas the Raw Data will be displayed The Standard Deviation and CV are automatically calculated for every multiple replicate standard control blank and sample group Note CV 100 Std Dev Mean If the protocol contains transformation formulas the results will be displayed in the Transform column If viewing statistics for a protocol that uses a stored curve the Y Axis Data will be displayed for the standards Interpreting Results e 7 31 Auto Sensitivity Results Auto Sensitryity 7 32 e Interpreting Results For fluorescence and luminescence readers KCjunior provides an Automatic Sensitivity Adjustment feature as an alternative to the more manual method of defining multiple filter sets with varying sensitivity settings See Defining Filter Sets under Defining Protocols for more information If this feature is enabled KCjunior instructs the reader to perform several pre readings on the microplate In general these readings are performed on user specified wells using incremental sensitivity settings Each increase in sensitivity rais
157. he Well ID template before defining dilutions transformations or sample IDs User s Guide to KCjunior Defining Default Well IDs Well IDs are used to identify the four well types supported by KCjunior Blank Standard Sample and Control Common IDs for these well types are BLK STD SMP and CTRL respectively Default Well IDs are defined within the System Configuration dialog found under Setup Configuration Define these IDs before creating any templates For more information see Setting Up the System System Configuration in Getting Started Assigning Well Identifiers Well information such as type location replicates and blanking method is defined in the Well ID Template Protocol Definition x General Information Read Method Template Curve Cutoffs Reports WelllDs C Dilutions Transformations C Sample IDs SMP13 SMP21 SMP37 SMPOB SMP14 SMP22 SMP38 SMP07 SMP15 SMP23 SMP39 SMPO8 SMP16 SMP24 SMP40 E smPo1 sMpo s SMP09 SMP17 SMP17 SMP25 SMP41 F smPo2 sMPo2 SMP10 SMP18 SMP26 l SMP42 SMP11 SMP19 SMP27 SMP43 SMP12 SMP20 SMP28 SMP44 Well Type Selections m Replicates Direction m Sample Number Sample 2 45 Blank Method C Down Actoss M Auto Increment Full ae m Map Direction Down C Actoss Cancel Designing a New Well ID Template To design a Well ID Template
158. he formula If blank subtraction is performed on the Raw Data or the Result value the Blanked Data value is used in the formula gt Click the Advanced Options button to change the Test or Reference Wavelength values or to change the Constant Value Pathlength Correction Options Dialog EI Test Wavelength nm 377 Reference Wavelength nm 900 Cancel Help r Constant Values Use Default Settings Absorbance of water at 1 0 cm 977nm 900nm 0 18 C Other Value i The valid range for the Test and Reference Wavelengths fields is 200 999 nm To define a Constant Value other than 0 18 for use with calculating pathlengths click Other Value then enter the desired number Click Use Default Settings to reset the Test and Reference Wavelengths to 977 and 900 nm respectively to reset Other Value to 1 and to reselect the use of the Absorbance of water at 1 cm value 5 18 e Defining Protocols User s Guide to KCjunior User s Guide to KCjunior Example 1 A protocol specifies the following gt MultiWavelength read method type gt Microplate read at 405 nm gt Pre read an empty plate at 405 nm gt Pathlength Correction is enabled gt The default value of 0 18 represents the absorbance of water at 1 cm 977 900 nm The empty plate is read at 405 nm to obtain the blanking absorbances for each well The sample plate is initially read at the Test and Reference Wavelengths Well Al re
159. he report also contains the mean standard deviation and coefficient of variation for each Sample ID Well ID The Matrix Report contains well specific information in a matrix corresponding with the plate geometry The contents of this report can be customized In the Options box highlight up to 8 items to appear on the report then click the Add button The selected item s will appear in the Selections box Available options depend on the current protocol settings See Selecting Options for the Matrix Report on the next page for more information To remove an item from the Selections box highlight the item and click the Remove button To insert a reporting option between items in the Selection box highlight the item in the Selection box above which the new option will be inserted highlight the data set in the Options box then click Add User s Guide to KCjunior gt The Curve Report contains the graphical representation of the standard titer or spectral scan curve as appropriate for the protocol 5 Select OK to close the protocol and retain the new settings or click another tab to define additional information e The contents of some of these reports can be further customized for greater flexibility with data presentation e Appendix A of the User s Guide to KCjunior contains samples of each report type e Most reports require that well identifiers be assigned in the template The Raw Data Report
160. heck the Cutoff dialog in the protocol to ensure that cutoffs are defined correctly e Ifa cutoff references a well ID ex STD02 this error may indicate that the actual mean value for the well ID greatly varies from its expected value DIV 0 DIV O represents an attempt to divide by zero Suggestion e This error is most likely to appear in the Statistics dialog after an unsuccessful attempt at a CV calculation Click the Statistics button to view the results Any DIV 0 warnings will appear in the CV column Error during auto sensitivity This message indicates a failure during the auto sensitivity adjustment process The most likely reason is an error during the calculation of the signal to noise ratio Review the Auto Sensitivity selections in the protocol e Ensure that the Low Test and High Wells are assigned correctly gt The Low Well s are usually blank wells buffer wells zero standards or negative controls gt The Test Well s are usually low standards or low positive controls gt The High Well s are usually the highest standards or high positive controls e The Base Value should be greater than zero The default setting is 100 e If the High Value is set too low the Low Test and or High Wells can overrange before two valid signal to noise ratios can be calculated Troubleshooting e 10 7 10 8 e Troubleshooting Grayed numbers indicate a suppressed standard If in the Results displa
161. however can be printed even if no well ID s are assigned Selecting Options for the Matrix Report The options available for printing depend on the current protocol settings and can include some or all of the following e Well ID e Raw Blanked and or Corrected Data gt Endpoint and Kinetic absorbance reads The options to include raw or blanked data in the Matrix Report are presented as Raw Data or Blanked Data gt Endpoint and Kinetic fluorescence luminescence reads There are multiple Raw and Blank options available for the Matrix Report one for each filter set defined For example if the Read Method options specify 3 filter sets and there are blank wells defined in the template the selectable Matrix Report options include the following Raw 1 Raw 2 Raw 3 Blank 1 Blank 2 Blank 3 Raw 1 represents the raw data values obtained with filter set 1 Blank 1 represents the values obtained with filter set 1 after blank subtraction and so on User s Guide to KCjunior Defining Protocols e 5 63 gt MultiWavelength absorbance reads An option is presented for each individual wavelength For example if the protocol specifies reading at 405 450 and 490 nm with blanking the options include the following Raw 405 Raw 450 Raw 490 Blank 405 Blank 450 Blank 490 If Read Empty Plate is enabled for a wavelength the Raw and Blanked data sets for that wavelength represent the
162. icactrtancramata si eunidmed N a 5 6 Detimin Protocol Parameters oseca a a a a A 5 8 Metinine Gencrallntormna oars asaen toe ieaeisds vorsaa epoacawiavaenen be teawctaenen eetawes 5 8 Read Method Par aime ters engeni a A 5 10 NEC ACES onea at tuidus casas outa eacuuadiindedad teoduadeeatadantadenoieinaetel ences dinaate 5 38 OS Tigi eerie ner enter ete Cate mvt ee een ete meter or ae ate neni ene nt net A net ae 5 55 OS ONS arse osetia T E Wiech san eone E cad atenet Wreessdsadnoone went ade 5 58 REPOT US ote cai bettas aaa tata atie Rena yt tenant T att et sonia 5 61 Reading Plates 6 1 CV CUNO Wonnie in Bettas Cecad ude dePaai ead meee Resend eee det uai A Deinead eee ead ouaaee 6 1 W oekine With Plates and Results 260 55 tow ais iets ian stent Mads cata sash ein taas aleve Saeed aceu a Sie Sauts 6 2 Wiieteis Plate Data Stred laara n a e EA 6 2 Opening an Existing Results Record nnneeeeeesssssssssssssseerrreressssssssssssseererressssssss 6 2 Generating a New Setor Plate Datars a a e a a a 6 3 Savine and losins Results Records ronnri A teee 6 4 Copying an Existing Results RECOM cisistsstrecunccenededss tus Gadisaseudentadunpinincedgstendds ieaaiess 6 4 Sharing Results Records and Databases ccccccccccccsnssssssseseseeeeeceeeeeeeeeeeaaeaeeeeeees 6 5 Miporin e AR SUG 55 tales ccahtcDainee septs a atsadeanta E R 6 6 ii e Contents User s Guide to KCjunior Redno a AUG irae oar satura sea ran ete a aati Baoan awanias
163. iguous items gt Check Delete after archive to permanently delete the item s from the current database after being copied to the archive database Click the Archive Curve s button The selected items are copied to the archive database and then deleted from the current database if Delete after archive is checked gt Ifa duplicate Curve Name is detected KCjunior does not overwrite the curve Utilities e 9 17 9 18 e Utilities Archiving Test Results When Reader System and Universal Plate Test Results are archived they are copied from the current Results Database into the Archive Results Database To archive test results L 2 Make sure that an Archive Results Database has been created Select Utilities Database Maintenance Archive System Test Results or Universal Plate Test Results The Archive Results Database Pathname dialog appears Archive Results Database Pathname x Results Database Pathname Ic KCjunior D atabase Archives Archive Results mdb Browse Cancel Help Enter the drive directory and file name of the archive database or use the Browse button to locate this information Click OK The Archive System Test Results or Archive Universal Plate Test Results dialog appears listing the test results stored in the current Results Database Archive System Test Results x Test Results Archive Test s 1175 99 2 54 26 PM 100413 11 5 99 2 58 32 PM 100413 Exit 11 5 99 3
164. ile name in the Export File field gt Example C KCjunior Results Export 01 txt gt Long file names are supported If no file extension is specified KCjunior automatically uses txt text file gt Alternatively click Browse to search the directory structure Locate and highlight the desired file name then click Open to select it or travel to the desired file storage location enter a new file name then click Open to create select it gt The file can be stored on a computer s hard drive on a network to which the computer is attached or on a floppy disk gt If the file name already exists KCjunior provides the option to overwrite the entire contents of the file or to append the new data to the end of the file Reading Plates e 6 19 6 20 e Reading Plates 7 Choose a Delimiter or define a new one gt gt KCjunior uses delimiters to separate individual values such as raw ODs calculated concentrations and transformation results Choose Tab or Comma or choose Other and enter a custom delimiter such as or Example If the comma delimiter is used exported Raw Data will resemble the following 1 235 0 267 0 541 1 749 0 232 2 418 0 784 0 154 1 982 0 247 1 583 3 148 2 961 0 235 1 287 1 768 0 336 0 234 0 984 2 514 1 674 3 992 1 852 0 235 Tip Ifthe file will be imported by another application check to see which delimiter s that application accepts 8 Click the Export button gt If
165. imits can be adjusted so that calculations are based on a subset of the total number of measurements 1 From within any kinetic results display click the Calculation Options button to open the Kinetic Calculations dialog Kinetic Calculations Dialog Read Limits Total Reads TEA First Read Help Last Read Slope Calculation Single Slope Points 7 Slope Masimum Slope Unit of Measure m min 2 To change the number of points for inclusion in the kinetic calculations enter a new First Read and or Last Read value then click OK The kinetic results redisplay showing only the points in the new calculation range The Maximum Slope automatically recalculates based on the new calculation range Altering the Maximum Slope Calculation The Maximum Slope is initially calculated based on the total number of kinetic points single slope The number of successive points for inclusion in the slope calculation can be adjusted 1 From within any kinetic results display click the Calculation Options button to open the Kinetic Calculations dialog Slope Calculation Single Slope Points Slope E Masimum Slope Unit of Measure mOD min m zec mOO min m hour 100 zec ODmin hg OD hour 7 8 e Interpreting Results User s Guide to KCjunior User s Guide to KCjunior 2 Deselect Single Slope Enter a new value in the Points Slope field 4 Optional Select a Maximum Slope Unit of Me
166. ined wavelength unless the WV function is used to specify a different wavelength e For multiple Filter Set reads Plate Formulas are applied to the Raw or Blanked Data for the first defined filter set unless the FS function is used to specify a different filter set See Defining Transformations Formula Syntax in Defining Protocols for more information on the WV and FS functions User s Guide to KCjunior Interpreting Results e 7 29 Statistics 7 30 e Interpreting Results Within each results display the Statistics button is available if a Well ID Template is defined in the protocol When the Statistics button is clicked KCjunior automatically calculates and displays the mean standard deviation and coefficient of variation of every replicate group 14 351 15 677 Hp 15 290 15 129 gt Well Types Samples 41 286 41 179 C Standards 41 428 C Blanks Controls 41 298 i 48 902 48 460 48 758 48 707 31 341 31 889 30 978 31 407 43 434 43 614 43 636 e Click the Samples Statistics or Controls button to see a different set of statistics or click OK to return to the results display e For MultiWavelength reads the Raw Data column contains the Read or Result if an empty plate was read values for the first defined wavelength The Blank Data column contains the blanked data for the first defined wavelength e For multiple Filter Set reads the Raw an
167. ing Open a dialog containing information about the plate such as ID description status and creation date and time Save the results record Save the open results record under a new Results ID Import results records from a stored Results Database into the one currently in use Export plate data to a text file or directly into a Microsoft Excel spreadsheet Print results according to reporting options specified in the Protocol See what the reports look like on the screen before printing Getting Started e 3 3 Print Setup Change the printer paper size or source or page orientation Exit Close KCjunior If a results record is open and unsaved KCjunior provides the option to save before exiting Protocol Menu The Protocol Menu contains functions for creating modifying saving and importing assay protocols Protocols Utilities Setup Nem Protocol Open Protocol Close Protocal Modify Protocol Save Protocol Save Az Inport Protocols New Protocol Create a new protocol Open Protocol Open an existing protocol Close Protocol Close the open protocol If the protocol has not yet been saved KCjunior provides the option to save before closing Modify Protocol Modify the current open protocol Save Protocol Save the open protocol Save As Copy the current open protocol to a new protocol with a different name Import Import protocols from a stored Protocol Database Protocols into
168. ining Protocols User s Guide to KCjunior Read Method Dialogs for Fluorescence Luminescence Readers Protocol Definition General Information Read Method Template Curve Cutoffs Reports Read Method Type Endpoint x Help Chemistry Fluorescence Luminescence ___GetReaderFiters Filters Validate Read Method Filter Sets 4 z Select Cartridges Excitation 360 40 485 20 s6040 485720 Emission 60740 528 20 ac0 40 528 20 Optics Position Top Top v Bottom Bottom Sensitivity ho 150 ho iso Options Options Options Options Plate Geometry 6x8 F Read Al F8 IV Eject between filter set reads Tl Monitor Well Monitor Well Parameters Cancel e During an Endpoint Read the plate is read from one to six times depending on the number of filter sets specified One Relative Fluorescence Unit RFU or Relative Luminescence Unit RLU value is reported for each well for each filter set Protocol Definition General Information Read Method Template Curve Cutoffs Reports Read Method Type Kinetic Hep Chemistry Fluorescence Luminescence ___GetReaderFilters Validate Read Method Filter Sets 2 z Select Cartridges Excitation 360 40 485 20 Kinetic Options Emission e0740 528 20 Kinetic Reads fo ooo Optics Position Top Top Kinetic Interval sec z5 I Fastest Interval Sensitivity fioo 150 Total Read
169. initialize the dispenser 1 Select Utilities Dispenser Control Panel 2 Click Initialize Dispenser The dispenser s syringe drive will User s Guide to KCjunior move to its home position and its sensors will be verified Utilities e 9 5 Priming the Dispenser The dispenser s syringe and tubing must be fully primed with the desired fluid before plate reading can begin In addition the FLx800 operator s manual contains decontamination procedures that require the dispenser to be primed Important A special priming plate Bio Tek part number 7142002 must be placed in the carrier prior to priming To prime the dispenser 1 Fill the supply bottle with the desired fluid Place the priming plate on the carrier In KCjunior select Utilities Dispenser Control Panel Click Prime Dispenser Enter the desired Prime Volume from 5 to 5000 ul oe Ne et See Select a Prime Dispense Rate from to 4 gt Note With each rate selection the corresponding ul sec value is displayed 7 Click Start Prime to begin the prime Purging the Dispenser KCjunior provides a utility for purging fluid from the dispenser by pumping the fluid in reverse back into the supply bottle Important The tip priming trough must be placed in the carrier prior to purging During this process the carrier is positioned so that the tip priming trough sits under the fluid injector head To purge the dispenser 1 Ensure that the tip priming
170. install ECjunior in the following directory To install in this directory click Hest To install in a different director click Browse and select another directory Click Cancel to exit the Setup program Destination Directory CAK Ciunio Browse Cancel 13 The default location for the software is C KCjunior Click Next to accept the default location or click Browse to specify a different location e Ifyou click Next an informational dialog appears Information Install will now ask for the location where you would like to store the PROTOCOL database IF You are unsure where it should be use the default location 14 Click OK to acknowledge the information 2 8 e Installing the Software User s Guide to KCjunior A protocol contains instructions for controlling an attached microplate reader and information for analyzing plate data Protocols are stored in the Protocol Database This database is typically stored on a computer s hard drive or on a network to which the computer is attached User s Guide to KCjunior The Choose Protocol Database Location dialog appears Choose Protocol Database Location Select 4 directory where you would like the Protocol database to reside To install in this directory click Next To install in a different directory click Browse and select another directory Click Cancel to exit the Setup program 15 The default Protocol Database Location is the
171. ion SD The standard deviation is a measure of how widely values are dispersed from the average value the mean In formulas standard deviation is represented by SD Template In each KCjunior protocol four unique templates can be designed to indicate location and related information for Well IDs Dilutions Transformations and Sample IDs Transformations Transformations are user defined formulas applied to plate data for analysis or for data transformation before calculating concentrations or cutoffs Two types of transformations can be defined within one protocol e A transformation formula is performed only on the well s to which it is assigned If Transformation Formulas are applied the results serve as the basis for curve generation and cutoff evaluation e A plate formula is automatically applied to every well on the plate that has been assigned a Well ID Well Identifier KCjunior supports four different types of wells Blank Standard Sample and Control To accommodate a variety of applications KCyunior provides the flexibility to customize references to these wells These customized references are called Well Identifiers 12 4 e Glossary User s Guide to KCjunior Index 2 Parameter curve 5 56 4 Parameter curve 5 56 A Aborting a read in progress 6 9 6 11 10 9 Add to List button 5 48 5 49 Addition in a formula 5 50 Adjusted value 7 37 Algorithm for auto sensitivity adjustment 7 33 A
172. ion 3 parameter changes to take effect on an EL311 the reader must be turned off on after any changes are made oe An Select the appropriate Reader Type before defining protocols even if the reader is not actually connected to the computer Click Get Basecode Version to retrieve the attached reader s basecode software part number and version number User s Guide to KCjunior Ensure that the microplate reader is connected to the computer on which KCjunior is loaded as described in the previous section From the reader s front panel if the reader is equipped with one find the Baud Rate Data Bits Stop Bits and Parity information Refer to the reader s user manual for assistance if necessary Note the current settings for each parameter Make the following changes if necessary gt If the Baud Rate is currently set to 1200 change it to at least 2400 gt If Data Bits or Char Bits is currently set to 7 change it to 8 Perform a reader system test if any changes were made above Important Return the reader s display to the main menu Start KCjunior Within KCjunior s main screen select Setup Reader 1 or Reader 2 The Reader Setup dialog appears Reader Setup 1 Reader Type D Cancel Help Com Fort Eaud Rate Get Comm Defaults Data Bits Test Communications Parity Get Basecode Version Stop Bits EOT Character 26 0014 Select a Reader Type from the dro
173. ion information can be viewed under the Template tab Raw Data Blanked Data Concentrations Template Comments Warnings Standard Curve e The Well ID or Sample ID and corresponding concentration dilution value are displayed for each well e Well IDs are used to identify the four well types supported by KCjunior Blank Standard Sample and Control User s Guide to KCjunior Plate Formula Results Plate Formula Results are displayed if a Plate Formula is defined in the protocol Raw Data Blanked Data Concentrations Template Plate Formula Results Comments Warnings 0639 osso 1 159 l 4 1 183 188 191 194 1 740 a79 1741 4 1 743 743 1 743 1 77 1 1 7711 1 772 1 774 1 781 782 1 786 1 1 788 1 789 1 790 1519 a519 1520 1520 asal 1 1521 1522 1 522 1 524 1524 1 525 15235 1 1526 1 526 1 526 t 1 527 528 528 528 528 2583 2585 2587 2 593 596 2 602 2 603 2 605 Standard Curve e ach well contains the result of the Plate Formula defined in the protocol e For Endpoint reads the Plate Formula is based on the OD RFU or RLU values e For Kinetic reads the Plate Formula is based on the Maximum Slope values e For MultiWavelength reads Plate Formulas are applied to the Raw or Blanked Data for the first def
174. ion specific to that plate such as Results ID Plate ID read status and raw plate data values Dilution The amount by which a sample is mixed with a solution to bring the specie of interest into the operating range of the analysis Endpoint Read During an endpoint absorbance read every well on the plate is read with one single or two dual wavelengths e One optical density OD value is reported for each well During an endpoint fluorescence or luminescence read the plate is read from one to six times depending on the number of filter sets specified e One Relative Fluorescence Unit RFU or Relative Luminescence Unit RLU value is reported for each well for each filter set Extrapolation KCyjunior provides the option of displaying extrapolated concentrations unknown values on the Y axis which are outside the range of the standards On data displays and reports extrapolated concentrations are enclosed by parenthesis for example 20 345 12 2 e Glossary User s Guide to KCjunior Kinetic Read During a kinetic absorbance read every well on the plate is read a defined number of times with one single or two dual wavelengths e One optical density value is reported for each well for each plate read In addition a graph showing OD vs Time is reported for each well During a kinetic fluorescence or luminescence read the plate is read a defined number of times using one filter set e One Relative Fluoresc
175. is initially reported in this format reporting the raw values for each well of Kinetic Read 1 Filter Set e Each well contains the RFU or RLU value calculated for the the Herseta first filter set defined in the protocol If two filter sets are defined use the Filter Set drop down menu to choose a different filter set Filter Set 2 e The word Dispense in a well indicates that a dispense or dispense related activity occurred well mode only e To view the results for a different kinetic read enter the desired read number in the Kinetic Read field or click the up down arrow buttons 7 14 e Interpreting Results User s Guide to KCjunior Kinetic Curves Dispense Read Kinetic read results are shown below in the Kinetic Curve format Raw Data Template Kinetic View Options C Raw Data Kinetic Curve e Each well initially displays a graph of the data points corresponding to the RFU RLU readings taken over time e To enlarge the view of the curve in any well click once on the desired well Well Kinetic Curve Dialog Ea gen Help Print 7 Copy Interval First Interval Interval 1 Second Interval C Interval Dispensing Alllntervals x Axis Scale C 001 sec 40 50 60 C 01sec 1 Seconds 1sec C 1sec gt Place the cursor over a data point to see its RFU RLU value and reading Time gt Dependi
176. junior 3 Click Run KCjunior in Trial Mode if you have not yet obtained a password The KCjunior product information dialog appears KCjuntor 4 Click OK to continue An initialization message appears then after a few moments the KCjunior main screen appears User s Guide to KCjunior Installing the Software e 2 13 The reader does not need to be connected to the computer to specify a Reader Type Select the reader type PowerWavexX for any PowerWavexX reader model other than the PowerWave XS 2 14 e Installing the Software 5 The Reader Setup dialog automatically appears the first time KCjunior is started following software installation Reader Setup 1 Reader Type Cancel Help Com Port Baud Rate Get Comm Defaults Data Bits Test Communications Parity Communications Settings Stop Bits EOT Character 26 001 4 If this dialog appears select a Reader Type The Reader Type represents the name of the reader to be controlled by KCjunior Click z to drop down the list then click on the desired reader Important notes gt Select the appropriate Reader Type before defining protocols even if the reader is not actually connected to the computer gt Choose carefully it is important to select the correct model For example be sure to select ELx808I EL808I for an 808 with the incubation option gt Select PowerwaveX for any PowerWaveX model other
177. k Print to print the results or OK to close the results Test results are automatically saved in the Results Database 10 Repeat steps 4 8 for the remaining wavelengths 11 Click the Exit button to return to the main screen Setting Up a New Data Sheet A data sheet contains exact spectrophotometric calibration data associated with a neutral density filter at 1 of 8 possible wavelengths To create a set of data sheets one for each wavelength for use with the Universal Plate Test 1 Click New Data Sheet from the Universal Plate Test dialog The Data Sheet dialog appears Universal Plate Test Data Sheet Dialog Universal Plate 9000547 Universal Plate 7260522 7 filter Cancel Serial Humber f 29379 Help Wavelength nm 405 Location 0D User s Guide to KCjunior Utilities e 9 11 9 12 e Utilities Select a Universal Plate gt 9000547 does not have a filter in the C6 location It is the standard test plate that can be used on any Bio Tek reader If this plate is selected the Peak Wavelength Test will not be performed gt 7260522 has a filter in the C6 location for the Peak Wavelength test Itis the test plate that is used on readers that support spectral scanning Enter the test plate s Serial Number Enter a Wavelength or select one from the drop down list Refer to the calibration data table that came with the test plate to determine the expected OD for each test well at the select
178. l Database Pathname for retrieval and storage e The Protocol Database Pathname contains the name and location of the current protocol database Multiple protocol databases can be stored on a computer or on a network but there can only be one current database gt Example pathname C KCjunior Database Protocol mdb e Select Setup Configuration Pathnames to view or modify the current protocol database pathname See Pathnames in Setting Up the System for more information Opening an Existing Protocol To open an existing protocol begin by clicking the Open Protocol button in the KCjunior main screen selecting Open Protocol from the Protocol Menu or by selecting a name from the protocol drop down menu in the toolbar The Open Protocol dialog appears Open Protocol Dialog Protocols Cancel Help e This dialog lists the names of all protocols available in the currently configured Protocol Database e To open a protocol highlight the desired name in the list then double click the mouse button or click the Open button User s Guide to KCjunior Hew Protocol Set the appropriate Reader Type before defining a protocol See Reader Setup in Getting Started for more information User s Guide to KCjunior gt If KCjunior is configured for use with two readers When a protocol is opened KCjunior attempts to determine which reader best supports the protocol and then sets it as the Current Reader
179. lable gt Discard the out of range value If Discard Out of Range Values is selected the blank well is reported as OUT and is excluded from the calculation of the blank mean gt Retain the out of range value If Discard Out of Range Values is not selected KCjunior automatically sets the raw OD RFU or RLU value to the Minimum or Maximum Blank Limits value as appropriate This new value is displayed inside brackets 0 010 and is included in the calculation of the blank mean Set the Displayed Decimal Places value to 0 1 2 or 3 This value overrides the setting under Setup Configuration Reporting Options Values such as optical density measurements transformation results and concentrations will be reported with the number of decimal places defined in this field unless Scientific Notation is enabled Defining Protocols e 5 9 7 Optional Check Scientific Notation Raw Data Blanks to report Raw and Blanked data in scientific notation Check Scientific Notation Transforms Concentrations to report Transformation results and calculated Concentrations in scientific notation If Scientific Notation is not enabled KCjunior automatically displays a very small number like 0 00004 as 0 000 instead of as 4e 05 Read Method Parameters The configuration of the Read Method parameters controls plate reading The availability of individual Read Method parameters depends on the current reader type and can include read method ty
180. late Curve Cutoffs Reports Read Method Type Endpoint Help Primary Wavelength nm 405 v Get Reader Wavelengths Validate Read Method Calibrate Reader Reference Wavelength nm 630 z Number of Plates fi Plate Geometry ext 2 x Read Al H12 Delay Before Read sec 20 Monitor Well v Monitor Well Params Shake Method Shake First Read as Intensity Variable T Incubation v Duration Temperature 37 isonthuaus Shake e During an Endpoint Read every well on the plate is read at one or two wavelengths One optical density OD value is reported for each well Protocol Definition General Information Read Method Template Curve Cutoffs Reports Primary Wavelength nm 450 Reference Wavelength nm 32 Kinetic Reads hoo o Kinetic Interval sec j5 J Fastest Interval Plate Geometry fies Read Al P24 Delay Before Read sec fio Monitor Well E Help Get Reader Wavelengths Validate Read Method Calibrate Reader Read Method Type Total Read Time Shake Method Shake Every Read Intensity Incubation Duration Temperature Continuous Shake i Cancel e During a Kinetic Read every well on the plate is read a defined number of times at one or two wavelengths One optical density value is reported for each well for each plate read A graph showing OD vs Time is reported for each well Defining Protocols e 5 11 Read Method Dialogs for Ab
181. length for each well 2 Calculates the corrected absorbance for each well 3 Displays the corrected absorbance for each well under the Pathlength Correction results tab gt KCyjunior calculates each well s pathlength using the following formula A977 nm Agoo nm Well A977 nm Agoo nm 1 0 cm water The well is measured at 977 nm and again at 900 nm then the difference is calculated The result is divided by the absorbance of water at 1 cm 977 nm and 900 nm represent default Test and Reference Wavelengths respectively These wavelengths can be changed under Advanced Options KCjunior uses a default value of 0 18 for the absorbance of water at 1 cm This default value can be overridden by changing the Constant Value under Advanced Options Alternatively click Use Bio Cell to calculate the absorbance of water at 1 cm value using Bio Tek s Bio Cell plate Set the Location of the cuvette in the plate Defining Protocols e 5 17 gt KCjunior calculates the corrected absorbance for each well according to the following formula Awavelengthwell pathlength of well The measurement taken at the user defined wavelength is divided by the calculated pathlength The Raw Data value of the well is used in the correction formula unless an empty plate is read or blank subtraction is performed If an empty plate is read the difference or Result in the Raw Data readings between the full and empty plate is used in t
182. lengths 3 Optional For any defined wavelength check Empty Plate to perform two reads at the corresponding wavelength the first read on an empty plate and the second on the sample plate KCjunior automatically subtracts the value of each well of the empty plate from the corresponding well of the sample plate User s Guide to KCjunior The Empty Plate can represent an empty dry plate or a plate filled with solution for blanking Pathlength Correction requires the 5 and 6 filters to be set either to lt None gt or to the Test and Reference Wavelengths respectively The pathlengths can be included on the Matrix Report See Selecting Options for the Matrix Report at the end of this chapter User s Guide to KCjunior For example examine the Wavelength setup shown below Read Method Type Multiwavelength Wavelengths nm 340 W Read Empty Plate 405 W Read Empty Plate 490 Read Empty Plate The empty plate will be read at 340 nm and 405 nm The sample plate will be read at 340 nm 405 nm and 490 nm Optional Check Use Pathlength Correction to normalize absorbances to 1 cm This option is only available for readers that support the MultiWavelength read method and wavelength values up to 999 nm Pathlength Correction M Use Pathlength Correction M Use Bio Cell Location Ad T Advanced Options gt If Pathlength Correction is enabled KCjunior 1 Calculates the path
183. leshooting section for specific information on data reduction comments and warnings 6 16 e Reading Plates User s Guide to KCjunior User s Guide to KCjunior Selecting a Filter Wheel Cartridge Depending on the prompt options settings in the SetupIConfiguration Filter Wheel Configuration dialog the Select Filter Wheel Cartridge dialog may appear applies to FLx800 reader only Select Filter Wheel Cartridge Dialog Excitation Filter heel Ex Cartridge 1 View Cancel E mission Filter Wheel Help Em Cartridge 1 WIE Excitation 360 40 485 20 E restr 460 40 530 20 Select cartridges that support the curent read and place them in the reader Press OF when ready This dialog shows the Excitation and Emission filters required by the protocol The selected filter wheel cartridges must contain filters to support the protocol 1 Using the drop down menus select the desired Excitation and Emission Filter Wheel cartridges gt The contents of Excitation and Emission filter wheel cartridges are defined under Setup Configuration 2 After selecting a cartridge clicking View displays the cartridge s contents gt Click Cancel to return to the Select Filter Wheel Cartridge dialog 3 Click OK to save the changes gt If the selected cartridge s do not contain the necessary filters to support the current protocol either select a different cartridge or modify the protocol Reading Plates e 6 1
184. ll With each reading the sensitivity gain of the photomultiplier is increased The target well is monitored to ensure that it and hence the assay does not exceed the defined High Value Measurements stop when the target well reaches the High Value The sensitivity that produced the RFU RLU closest to the High Value without exceeding it is chosen for the microplate read To enable Automatic Sensitivity Adjustment and define the pre reading parameters for the Scale to Low High Range option Note For any of the following parameters enter a single well location ex Al or a rectangle of well locations ex B1 C2 1 Specify the Low Well Range to define the well s representing the expected Base Value see step 4 gt Low Wells are usually blank wells buffer wells zero standards or negative controls and are expected to yield a low RFU or RLU gt This range must not overlap the Test Well Range 2 Specify the Test Well Range to define the well s to be used in conjunction with the Low Wells to determine signal to noise ratios gt Test Wells are usually low standards or low positive controls and are expected to yield a mid range RFU or RLU gt For best results designate as the Test Wells those wells on the plate generating a signal at least 50 greater than the Low Wells gt This range must not overlap the Low Well Range 3 Specify the High Well Range to define the well s representing the expe
185. llow pathname selection at program start 4 12 Ambiguous curve 12 1 Apply to all wells 5 47 5 48 Apply to selection 5 47 5 48 Archive Archive protocol database 9 15 Archive results database 9 15 Archiving results records and protocols 9 15 Archiving stored curves 9 17 Archiving test results 9 18 Creating archive databases 9 15 Auto sensitivity adjustment Algorithm 7 33 Base value 5 30 Defining the pre reading parameters 5 28 High value 5 30 High well range 5 29 Low well range 5 29 Overview 5 28 Test well range 5 29 Use result for all filter sets 5 30 Viewing results of pre readings 7 32 Average standards 5 57 12 1 B Backup procedures 9 14 Bandwidth Defining in a filter set 5 25 Warnings 5 25 User s Guide to KCjunior Barcode scanning 4 10 6 11 Basecode version 4 3 Base value 5 30 Baud rate 4 3 4 4 Bio Cell tm 5 17 7 18 Bio Tek contacting 1 3 Blank Data 7 5 7 9 7 16 Blank limits setting 5 9 Blanking method 5 39 5 41 Blanks defining 4 5 5 38 Bottom optical probe 5 25 C Calibrate before first read 5 37 Calibrating the reader 5 37 6 8 Chemistry 5 21 Closing a results record 6 4 Coefficient of variation CV 5 50 7 30 12 1 Colors changing 4 5 4 9 Communication Connecting the reader to the computer 4 2 Errors 6 17 10 2 Get communication defaults 4 4 Port 4 4 Reader system test 4 4 9 8 Testing communication 4 5 Comparison value OD 5 33 Computer Minimum system requirements 2 1 2 2
186. lphanumeric characters 5 Click the Read Plate button The Read Method dialog appears Read Method Read Method Type Endpoint v Help Primary Wavelength nm 405 _Get Reader Wavelengths ot Reader Wavelengths Validate Read Method Reference Wavelength nm E30 v Calibrate Reader Number of Plates fi Plate Geometry ext 2 x Read Al H12 Delay Before Read sec 20 Shake Method Shake First Read z OR F Intensity Variable x ncubation v Duration fio Temperature 37 i Cancel 6 Define the basic Read Method parameters See Defining Read Method Parameters in Defining Protocols for more information 7 Optional If the attached reader supports calibration click the Calibrate Reader button to calibrate the reader at the defined wavelength s 8 Click OK to continue with the read A second Read Plate dialog appears Read Plate Dialog Place the plate on the carrier Press OF when ready Cancel Plate ID Joong a 6 8 e Reading Plates User s Guide to KCjunior 9 Optional Manually assign a Plate ID to this plate gt For multiple plate endpoint absorbance protocols each individual plate can have its own Plate ID assigned This prompt appears before each plate is read 10 Place the plate on the carrier then click OK to initiate the plate read gt Ifthe attached reader is equipped with a barcode scanner and Use Reader s Barcode Scanner for Plate ID is
187. lts 7 29 Printing reports 8 1 Raw Data 7 1 7 4 Results records 6 2 Statistics 7 30 Template 7 28 Transformation Results 7 19 Viewing 6 24 Viewing statistics 7 30 Results database 2 10 Archive results database 9 14 Creating an empty database 6 5 Importing results 6 6 Pathnames 4 10 RESULTS MDB 4 10 Sharing results records and databases 6 5 Version 4 12 Results ID 5 20 6 2 6 8 6 9 6 10 Results record Copying 6 4 Deleting 9 19 Exporting data 6 18 ID 6 2 6 8 6 9 6 10 Importing 6 5 6 6 Opening 6 2 Saving 6 4 6 9 6 11 Sharing records and databases 6 5 RFU RLU 12 1 R square 12 1 S Sample curves 7 22 Sample interval 5 21 Sample names assigning 5 54 Sample protocols and results 2 15 Sample reports 11 1 Samples Defining 4 5 5 38 Identifying 5 54 Sampling 5 27 Delay before sampling 5 28 Delay between samples 5 28 Number of samples 5 27 Sampling windows 5 22 9 5 Saving a results record 6 4 6 9 6 11 Scanning a plate barcode label 4 10 6 9 6 11 Scientific notation 5 9 5 10 User s Guide to KCjunior Screen resolution 2 2 SD standard deviation function 5 52 12 1 Select cartridges 5 23 Sensitivity Auto sensitivity adjustment 5 28 Optimum sensitivity 5 26 5 28 Specifying within a filter set 5 26 Setup configuration 4 5 5 39 Setup Menu 3 6 Shake Continuous during kinetics 5 35 Defining parameters 5 34 Intensity 5 34 Method 5 34 Sharing protocols and databases 5 5 Sharing results records and databases 6
188. lts Database Pathname within his or her copy of KCjunior to point to the network database A results database can be copied to a diskette or CD and exchanged between users If the database has grown too large to fit on a diskette create an empty results database then copy only the desired results records into the new database 1 Select Utilities Database MaintenancelCreate Archive Results Database The Create Empty Results Database dialog appears 2 Inthe Save in field select a location for the new database then enter an identifying File name 3 Click Save to create the database and return to the main screen 4 Select Utilities Database MaintenancelArchive Results The Archive Results Database Pathname dialog appears 5 Enter the drive directory and file name for the newly created empty database 6 Click OK The Archive Results dialog appears listing the contents of the current Results Database 7 Select the record s to be copied Do NOT check Delete after archive 8 Click the Archive Results button The selected records are copied to the new database Results records can be imported from one database into another See Importing Results below for more information Suggestions gt Results records can be imported from a master results database stored on a network gt Results records can be imported from a results database stored on a diskette Reading Plates e 6 5 6 6 e Reading Plates I
189. mation A1 onoo Kinetic Curve A1 ee 0 000 Mask Well 41 g Mask All Reads 41 0 000 Unmask All Reads 41 0 000 See Simulation Data A1 eed 0 824 1 147 ae BA i 0 475 0 481 2 167 Accessibility to some Results Options depends on the protocol definition the current results display and the method of obtaining plate data For example Modify Simulation Data is not available if the plate data was retrieved from a microplate reader All Results Options are described below and on the following pages Plate Information Detailed Plate Information is available from within any results display To access this information 1 Highlight any well in any results matrix 2 Click the right mouse button The Results Options menu will appear 7 34 e Interpreting Results User s Guide to KCjunior 3 Select Plate Information The Plate Information dialog appears Plate Information Dialog Plate ID 008561 2 a as Plate Read Time 8 29 2000 2 03 14 PM Cancel Help Flate Status aK Read Method Reader Powerwavers Created By Karen B Temperature C nra Monitor well Elapsed Time zec fzo Flate Description gt The Plate ID is an optional identifier associated with the plate Itis entered at run time either manually or via a reader s plate barcode scanner The ID can be changed here if the result file has not yet been saved gt The date and time of the plate reading is displayed in the
190. med each well contains the delta OD value calculated by the reader less the average of the blank wells e For fluorescence and luminescence reads 1 to 6 sets of data are presented depending on the number of filter sets specified in the protocol Initially the RFU RLU values for filter set 1 are presented Use the drop down menu at the bottom of the screen to choose a different filter set User s Guide to KCjunior Blanked MultiWavelength Data Blanked Data results for a MultiWavelength read are shown below Raw Data Blanked Data Template 0 800 1 441 1 993 2 033 1 770 1 774 1 778 2 846 Statistics e Each well contains the OD value after blank subtraction e Use the drop down list to view Blanked Data for a different wavelength e If an empty plate is read for a particular wavelength the empty plate measurements are subtracted from the raw data measurements to generate the Result values e g 405 Result Blank subtraction is then performed on the Result values Pathlength Correction Pathlength Correction can be enabled to normalize absorbances to 1 cm Its use is controlled within a MultiWavelength protocol s Read Method dialog See page 5 16 for more information If Pathlength Correction is enabled KCjunior first calculates the pathlength for each well The absorbance in each well is then divided by the pathlength resulting in the corrected absorbance
191. meters set to their default values Click the Read Method tab Define Read Method Parameters The configuration of the Read Method parameters controls plate reading L A ee a User s Guide to KCjunior Click the Get Reader Wavelengths button to retrieve wavelength settings from the reader Select a Read Method of Endpoint Select any Primary Wavelength Leave all other parameters set to their default values Click the Template tab Getting Started e 3 13 3 14 e Getting Started Design Templates In each KCjunior protocol four unique Templates can be designed to indicate location and related information for well and sample identifiers dilution values and transformations Well IDs L oe en Select Blank from the Well Type Selections list then click on wells A1 and A2 Leave Blank Method set to Full Select Standard from the Well Type Selections list Set Replicates Direction to 2 and Across respectively Click and hold the cursor on well B1 Drag the cursor to well H2 and release Seven groups of Standards should be assigned in duplicate Select Sample from the Well Type Selections list Set Replicates Direction to 4 and Down respectively Set Map Direction to Across Click and hold the cursor on well A3 Drag the cursor to well H12 and release Twenty groups of Samples should be assigned in quadruplicate The Well ID Template should look like this Protocol Definition 9 Ge
192. mmended or you can choose to wait and restart it yourself Choose an option then click Finish KCyjunior is now installed on your computer User s Guide to KCjunior Installing the Software e 2 11 To create a KCjunior shortcut icon for your Desktop gt Open Windows Explorer Make the window small so you can see the Desktop in the background gt In the KCjunior folder click and hold the right mouse button on KCJrWin exe Drag the item onto the Desktop and release the mouse button gt Select Create Shortcut s Here You have 30 days from the date of installation to use KCjunior without a password 2 12 o Installing the Software To Start KCjunior 1 From the Windows Start menu access the Programs menu then the KCjunior menu Click on KCjunior Accessones Extensions HTML Help Workshop Internet Explorer INTERSOLY E Cjunior Leverage Host Services McAfee VirusScan Microsoft Reference Mk Reflection Eind StartUp is Help Wee ech Run Winzip Wie ftp Shut Down atl Microsoft Exchange 2 If you have not yet entered your KCjunior password the Registration Form dialog appears Registration Form Run E System ID OF Password Cancel Remaining Days 30 You have 30 days from the date of installation to use KCjunior without a password See Password Distribution and Software Registration on page 2 16 for more information User s Guide to KC
193. more information on the WV and FS functions Interpreting Results e 7 19 Concentrations Concentrations are reported if curve parameters are defined in the protocol and if the curve generated successfully Concentrations are Raw Data Transformation Results Concentrations Template calculated for every well on ee el the plate based on the standard curve and i 20 250 20 986 21 511 21 906 22 457 33 692 according to a certain 37 16631 as227 sania 34 400 34 472 34595 34 594 46 5211 46531 46 540 structure See Essential 46 579 46 584 47 099 47 144 47 187 47 228 47 267 Information in Getting 576 245 l i 39 744 4749 47 524 47550 39852 43187 47 625 Started for more 33 484 6180 37 255 33513 61 839 33 531 33 981 information 41 304 50 579 51 910 51 91 ai 42 656 37 366 34 063 50 620 41 381 34104 36 758 41 397 42 726 65 121 73 986 65 196 65 371 65 404 65 436 65 468 65 499 65 530 65 560 65 589 Statistics Standard Curve e Each well contains its calculated concentration e For Endpoint reads curves are based on the OD RFU or RLU values e For Kinetic reads curves are based on the Maximum Slope values e For MultiWavelength reads curves are based on the measurements at the first defined wavelength unless Transformation Formulas utilizing the WV function are applied to specify a different wavelength e
194. mporting Results Results records from one results database can be imported into another results database The Results Database that will receive the imported records is the one defined under Setup Configuration Pathnames To import results records from a stored database L Ze If a results record is currently open close it Select Results Import Results The Import Results Database Pathname dialog appears In the Results Database Pathname field enter the pathname drive directory and file name of the database containing the records to be imported Alternatively use the Browse feature to locate and select the database Click OK The Import Results dialog appears listing all records in the database gt Click the Results and Creation Date column headers to sort the records alphabetically or by date Use the mouse to select the record s to import gt Press the Shift key while using the mouse to select contiguous records gt Press the Ctrl key while using the mouse to select non contiguous records Click the Import Results button KCjunior imports the selected records gt Ifa duplicate record name is detected KCjunior does not overwrite the record User s Guide to KCjunior Reading a Plate Read Plate User s Guide to KCjunior A microplate can be read with or without pre defining or opening a Protocol A protocol contains information for the operation of the microplate reader as well as instru
195. n OD RFU or RLU value is enclosed in brackets 2 999 the raw data value fell outside the acceptable min max data range defined in the protocol and was automatically set to the upper or lower limit of this range as appropriate If a Raw data value contains a strike through 2 684 it has been masked or temporarily removed from calculations See Results Options at the end of this section for information on masking and unmasking wells A well containing DIV O indicates a failed attempt to divide by Zero A well containing asterisks indicates a well status error If the concentration in a well is enclosed in parenthesis 20 345 it was calculated through extrapolation If the concentration in a well is enclosed in curly braces 10 765 it is ambiguous An empty well indicates no result was calculated Possible reasons include gt Ifa protocol is open no Well ID was assigned to this location gt The Raw Data value is currently masked The Raw Data value is out of range OUT v gt Ifthe well is empty under Concentrations Extrapolation may be disabled User s Guide to KCjunior User s Guide to KCjunior value could not be calculated Possible reasons include gt The well references another well with a status of Well Read Error OUT Masked Divide by Zero Well Failure A calculation within or involving the well attempted an invalid logarithmic function is syntactically invalid refer
196. ne graph Adjusting the Wavelength Range Used for Calculations Spectral scan calculations initially include all measurements taken across the wavelength range as defined in the protocol The wavelength range can be adjusted so that calculations are based on a subset of the full wavelength range 1 From within the Spectral Scan results display click the Calculation Options button to open the Spectral Scan Calculations dialog 2 To change the range of wavelengths for inclusion in the spectral scan curve for all scanned wells enter a new First Wavelength and or Last Wavelength value then click OK The Spectral Scan results will redisplay The spectral curves will show only the points within the new wavelength range and the Peak ODs will be recalculated Curve Display Options Click the Calculations Options button to change the curve display The available curve display options include e Display Points only e Display Line only This is a point to point line shown without the individual data points e Display Points and Line This is a point to point line and the data points are displayed See the example below Plate Sample Plate 10 Wel SMPO1 41 Help Print Copy Options I Display Legend 200 220 240 260 280 Wavelength nm Peak OD 3 713 at 260 nm 7 12 e Interpreting Results User s Guide to KCjunior Raw MultiWavelength Read Data Raw Data results for a MultiWavelength read are shown
197. need to be connected to the computer to read plate data from a file Select Results Read Plate or click the Read Plate button in the main screen The Read Plate dialog appears Enter a Results ID using up to 32 alphanumeric characters The ID is optional in this dialog but it will be required later if the results are to be stored in the Results Database Optional Enter a Plate Description using up to 1024 alphanumeric characters Click the Read From File button A standard Open File dialog appears Locate and highlight the desired file then click Open KCjunior examines the file to determine certain characteristics such as read method wavelengths number of reads and kinetic interval if applicable If there are no discrepancies KCjunior opens the file and calculates the results If KCjunior is unable to determine one or more of these characteristics a Read File dialog appears requesting entry of the unknown values The Read File dialog contains as much information as KCjunior was able to retrieve from the file Any unknown values must be entered before results calculation can begin gt Ifa field is dimmed and inaccessible KCjunior requires no further input gt If the Read Method field drop down box is accessible choose Endpoint or Kinetic as applicable gt Ifthe Primary Wavelength field contains 0 enter a wavelength from 1 to 999 Reading Plates e 6 13 gt Ifthe Reference Wavelength field contains
198. neral Information Read Method Template Curve Cutoffs Reports WelllDs Dilutions C Transformations C Sample IDs SMPOS SMPOT SMPO2 SMPO3 SMPO ee tS STDO2 SMPO SMPO2 TE STD03 SMPO1 SMPO2 saan STDO4 SMP11 SMP12 SMP14 SMP19 SMP20 STDOS SMP11 SMP12 SMP14 SMP19 SMP20 STDO6 SMP11 SMP12 SMP14 SMP19 SMP20 Well Type Selections m Replicates Direction m Sample Number Sample x 4 21 Blank Method Down C Across M Auto Increment Full z Han SGhstan STDO SMP11 SMP12 SMP14 SMP19 SMP20 Map Direction Help C Down Across z Concentrations Cancel gt Clear any mistakes by selecting lt Clear gt from Well Type Selections and highlighting the well s that need to be changed Click the Concentrations button User s Guide to KCjunior Concentrations Concentrations can be assigned from within either the Well ID template or the Curve dialog Here you will assign a concentration value to each standard group Set the starting Concentration to 1 000 2 Click Factor and set the Factor Value to 2 3 Click the title cell of the CONC column Concentrations should be assigned as 1 2 4 8 16 32 and 64 4 Click OK Click the Dilutions radio button Dilutions In quantitative assays calculated concentrations are multiplied by the dilution values to produce the final concentration
199. ng error Error AF SU e Ifa reader error message is presented note the error code ex AF90 then refer to the reader s Operator s Manual for more information The manual should contain a table of possible error codes e KCyjunior will attempt to save any data that was retrieved from the reader before the error occurred The Plate Status will be updated with the reader error code To check the Plate Status select Plate Plate Information Communication Errors Reader Status Failure KCjunior is unable to communicate with the attached reader Suggestions e Select Setup Reader then click Test Communications KCjunior will provide appropriate messages for reconfiguring the communication parameters e Make sure that the proper Reader Type is selected under Setup Reader then run the reader system test e Ifthe reader just experienced a failure the reader system test must be run before plate reading can resume Reader Timeout Error KCjunior received no response from the reader Suggestions e The reader should be on and at its main display when communicating with KCjunior e Try to perform a reader system test from within KCjunior 10 2 e Troubleshooting User s Guide to KCjunior If you cannot communicate with the reader run the test from reader front panel If the test fails refer to the reader s user manual for assistance e If the error appeared while performing a kinetic read Close all unnecessary program
200. ng on the current Dispense Read parameters you can adjust the X Axis Scale and or select an Interval sampling window to view gt A dotted green line indicates the start of a dispense gt A green triangle indicates that a dispense or dispense related activity occurred well mode only User s Guide to KCjunior Interpreting Results e 7 15 Blanked Data Filter Set pa Excitation 465 20 Emission 530 20 7 16 e Interpreting Results Blanked Data represents Raw Data that has undergone blank subtraction The format of the Blanked Data results display differs based on the defined Read Method Endpoint Kinetic MultiWavelength or Spectral Scan Note To view Blanked data for a Kinetic or Spectral Scan read click the Raw Data tab then click the Blanked Data radio button under Kinetic or Scan View Options See Raw and Blanked Kinetic Read Data on page 7 5 or Raw and Blanked Spectral Data on page 7 9 for more information Blanked Endpoint Read Data Blanked Data results for an Endpoint read are shown below Raw Data Blanked Data Template 0 384 0 359 0 229 0 615 1 716 1 660 0 720 0 878 0 773 0 803 0 227 0 230 0 069 0 079 0 036 0 126 Statistics e Ifa single wavelength absorbance read was performed each well contains the OD measured at the Primary wavelength less the average of the blank wells e Ifa dual wavelength absorbance read was perfor
201. nm gt The Spectral Scan Curve initially contains all ODs read at all wavelengths as defined in the protocol gt The Peak OD and its corresponding wavelength display at the bottom of the graph gt Move the mouse cursor over a data point to see its exact OD and Wavelength values gt Click Print Copy to print the graph or to copy it to the clipboard gt Click Options to adjust the curve calculations and or the display options for this well Peak OD Wavelength Typical spectral scan results are shown below in the Peak OD Wavelength format Raw Data Template Comments Warnings Scan View Options r Calculation Options C Raw Data C Spectral Chart Spectral Chart C Blanked Data Peak ODAVavelength Spectral Chart _ e The peak highest optical density reading and its corresponding wavelength value are reported for each well e The Peak OD is initially based on the complete wavelength range as defined in the protocol If the wavelength range is adjusted the Peak OD is redetermined based on the new range User s Guide to KCjunior Interpreting Results e 7 11 e If blank subtraction was performed the Peak OD is automatically based on the Blanked ODs e Click the Calculation Options button to adjust the wavelength range used for calculations for all scanned wells e Click the Spectral Chart button to select up to 8 spectral curves to overlay in o
202. ns or cutoffs The basis for formula calculation may be Raw Data Blanked Data data normalized by Pathlength Correction or the Maximum Slope depending on the protocol definition See Essential Information in Getting Started to review the results calculation structure Results calculationT wo types of formulas can be defined within one protocol e Transformation Formulas These formulas are manually assigned to individual wells Results of these formulas serve as the basis for curve generation and cutoff evaluation e Plate Formulas These formulas are automatically applied to every well on the plate that has a Well ID assigned Results of these formulas do not serve as the basis for curve generation or cutoff evaluation To define Transformations 1 Ensure that Well IDs are defined appropriately in the Well ID Template 2 Access the Transformations Template Protocol Definition General Information Read Method Template Curve Cutoffs Reports C WelllDs Dilutions Transformations C Sample IDs ELK1 X BLE CTL1 IX ELKINCTL1 ELK1 100 X PLE 1 CX BLE Lit 4 SMPO1 SMPO1 SMP02 SMP02 PLE1100 X BLE 4 BLE 4 BLE 4 BLE SMPO1 SMPO1 SMP02 SMP02 4 BLE 4 BLE 4 BLE SMPO1 SMP02 SMP02 100 4 DLE 4 BLE 4 BLE 4 DLE SMPO1 SMPO1 SMP02 SMP02 X BLEAWVCTLI BLE A100 X IX ELKIM X BLK1 X BLK1 X BLE Lt SMPO1 SMPO1 SMP02 SMPO02 SMP02 X BLEUMCTLA BLE 100 X ELKIWIX ELKOH hf MPO2 SM
203. nsider the following Changes made to the Well ID Template may affect the Dilution Transformation and Sample ID Templates as well For example if all PC wells are removed from the Well ID Template any Transformation containing PC will become invalid and should be modified or removed Changes made to the default Well IDs may affect modifications to the Well ID Template When a Well ID Template is created the default Well IDs that were defined via Setup Configuration are assigned to template wells as illustrated below iS ee eee Sa le e ne went smeo KCjunior requires that within one template a single Well ID must be defined for wells of type Blank Standard and Sample Because of this it is useful to know 1f any default Well IDs have been changed before modifying an existing Well ID Template In the following example the user plans to add one more blank well to a template in a previously saved protocol In the time since the protocol was last saved the default well ID for wells of type Blank had been changed gt The original Well ID Template was created as shown above with two wells of type Blank with the JD BLK The protocol was then saved with this template setup gt Via Setup Configuration a change was made to the default Well ID for wells of type Blank they are now referred to as BLANK instead of BLK gt To add another Blank well Well Type Selection is set to Blank then well C1 is click
204. omultiplier KCjunior 1 prohibits the saving of a protocol with overlapping Excitation and Emission wavelengths defined or 2 provides a warning if the wavelengths are considered to be too close but does not prohibit the saving of the protocol Just prior to a plate read KCjunior communicates with the reader to see if its filter table contains all of the filter bandwidth combinations defined in the protocol Choose an Optics Position of Top or Bottom gt Not all fluorescence luminescence readers are equipped with two probes When a plate read is initiated KCjunior communicates with the reader to see if the selected Optics Position is supported If the reader is connected and is turned on click the Validate Read Method button to see if the currently selected Optics Position is supported Defining Protocols e 5 25 gt LL l _ e gt D LL oc Range of samples Optimum sensitivity Samples fall within the 0 to 99999 RFU RLU range Lowest values are above 0 RFU or LFU Range of samples Sensitivity is too low Some samples are reported as 0 RFU RLU RFU or LFU Range of samples Sensitivity is too high Some samples are reported as 99999 RFU RLU 5 26 e Defining Protocols Specify the Sensitivity setting of the photomultiplier unless automatic sensitivity adjustment will be used see next step The signal from the range of fluorescence and luminescence assays c
205. on Note the current settings for each parameter Make the following changes if necessary gt If the Baud Rate is currently set to 1200 bps change it to at least 2400 bps gt If Data Bits is currently set to 7 change it to 8 Return the reader front panel display to the main menu Perform a reader system test if any changes were made User s Guide to KCjunior Define and Test Communication Parameters It is important to set and test communication parameters before attempting to read a microplate L 2 3 4 5 Start KCjunior if it is not already loaded In KCjunior select Setup Reader Select the appropriate Reader Type Select the appropriate Com Port setting Enter Baud Rate Data Bits Parity and Stop Bits values to match those on the reader Click the Test Communications button If the test fails follow the directions provided by KCjunior then click Test Communications again If the test passes click OK to save the settings and close the dialog Create a Protocol A protocol contains instructions for controlling the reader and for analyzing data retrieved from the reader L 2 3 6 Click the New Protocol button in the main screen Enter a Protocol Name using up to 16 alphanumeric characters Set the Raw Data Limits Minimum and Maximum Values to 3 000 and 3 000 respectively Set the Blank Limits Minimum and Maximum Values to 1 000 and 1 000 respectively Leave all other para
206. one for the top probe and the other for the bottom probe Comparison of results among various sampling settings User s Guide to KCjunior Get Reader Filters Each filter is imprinted with its wavelength bandwidth combination User s Guide to KCjunior To define Filter Sets 1 Ensure that the Read Method Type and Chemistry settings are correct If the reader is connected to the computer and turned on click Get Reader Filters to update KCjunior s internal filter table with the reader s current settings Select the number of Filter Sets 1 to 6 for endpoint or 2 for kinetic Perform steps 5 through 8 for each filter set Select an Excitation filter and an Emission filter gt The Excitation filter selects a narrow band of light to which the sample will be exposed The Emission filter selects a band of emitted light to be measured by the instrument s photomultiplier Valid selections include a wavelength bandwidth combination in nanometers Plug to indicate the presence of a plug and Open to indicate the absence of a filter or plug If the desired filter cannot be found within the drop down list manually enter the wavelength and bandwidth values The correct format is wavelength bandwidth in nanometers For example 485 20 represents the 485 nm wavelength with a bandwidth of 20 nm This combination permits the transmission of light from 475 to 495 nm To prevent damage to the phot
207. or Defining Protocols Overview A Protocol contains Instructions for controlling the attached microplate reader Information for analyzing data retrieved from the reader or for analyzing data entered manually or read from a file User definable protocol features include Protocol name and description Minimum and maximum optical density limits Minimum and maximum relative fluorescence luminescence unit limits Reading method Endpoint Kinetic MultiWavelength Spectral Scan or Dispense Read and related parameters Fluorescence Luminescence specific parameters such as chemistry number of filter sets optics position and sensitivity Template design for gt Well IDs gt Dilutions gt Transformation and Plate Formulas gt Sample IDs Curve fitting parameters Cutoff criteria Reporting options Defining Protocols describes how to customize the properties of every feature listed above User s Guide to KCjunior Defining Protocols e 5 1 Working with Protocols Open Protocol 5 2 e Defining Protocols This section provides information for creating changing accessing storing and sharing protocols Where are Protocols Stored Protocols are stored inside a Protocol Database which is a file that is typically located on a computer s hard drive or on a network to which the computer is attached When opening or saving protocols KCjunior automatically refers to a user specified Protoco
208. ore To visit the site from within KCjunior select Help Bio Tek on the Web then click on Bio Tek Home Page KCyjunior automatically loads your browser and goes directly to the site User s Guide to KCjunior Installing the Software Read Me User s Guide to KCjunior Complete the following to get KCjunior up and running L Ensure that the computer on which KCjunior will be installed meets the minimum system requirements Remove the previous version of KCjunior from your computer if one exists Install KCjunior Contact Bio Tek to obtain a password to ensure continuous use of the software after the 30 day trial period Register your copy of KCjunior with Bio Tek to record your warranty and to receive information on upgrades and new products Connect your microplate reader s to the computer Configure the reader s and KCyunior so that they can communicate with each other Installing the Software covers steps through 5 See Chapter 4 Setting Up the System for information on connecting a reader to the computer and configuring the reader and KCjunior Installing the Software e 2 1 System Requirements For KCjunior to run successfully you will need the following e Intel 486 processor 33 MHz minimum Pentium 66 MHz or greater is recommended e Microsoft Windows 95 98 2000 NT or XP operating system e 8 MB of RAM minimum 16 MB or more is recommended e 20 MB of available hard drive space for th
209. ormation plate and cutoff formulas Each Well ID can contain 2 to 5 alphanumeric characters with no spaces Note Changes to default Well IDs may affect future modifications to existing Well ID templates See Modifying an Existing Well ID Template in Defining Protocols for more information 4 6 o Setting Up the System User s Guide to KCjunior User s Guide to KCjunior gt Usage example If an assay requires the use of Calibrators to generate the standard curve set the Selection name and Well ID for Standard wells to Calibrator and CAL respectively as shown below Standard Calibrator EAL With these settings KCjunior generates the standard curve based on the ODs measured in the wells assigned as Calibrator For more information see Designing Templates Well ID Templates in Defining Protocols 3 Click another tab to configure additional system parameters or click OK to save these settings close the dialog and return to the KCjunior main screen Reporting Options KCjunior provides several configurable reporting options including precision reader temperature and special graph options To configure Reporting Options 1 Select Setup Configuration Click the Reporting Options tab The Reporting Options dialog appears KCjunior Configuration WellIDs Reporting Options General Pathnames Filter Wheel Cartridges m Precision Help Displayed Decimal Places 3 T T
210. ormation on upgrades and new products Before you contact Bio Tek record the System ID from KCyjunior s opening screen System ID Password Cancel a l Help Remaining Days 14 The System ID is an identifier that is unique to your computer Bio Tek will create a password for you based on this number When you receive your password enter it in the Password field shown above then click OK e This password only needs to be entered once KCjunior will not ask you for it again e If you need to reinstall KCjunior on the same computer this password will still be valid e This password will only work with your computer Contact Bio Tek with any questions User s Guide to KCjunior User s Guide to KCjunior Obtaining a password and registering the software online If KCjunior and your Internet browser are on the same computer launch KCjunior and select Help Bio Tek on the Web Register KCjunior Get Password Help Topics a Cl j ta Bio Tek on the web Bio Tek Home Page Register KCiunior Get Password About KCjunior KCjunior will launch your browser and go directly to Bio Tek s website A Password Distribution Software Registration form will appear Follow the directions to fill out and submit the form If your computer is not connected to the Internet enter this address into your browser http www biotek com labcore htm Click the Software Registration link to access the form O
211. orts e 8 3 8 4 e Printing Reports User s Guide to KCjunior Utilities Overview User s Guide to KCjunior Special reader and database maintenance services are accessible through the Utilities menu including Incubation Control reader dependent Carrier Out In reader dependent Dispenser Control Panel Reader System Test Universal Plate Test Database Maintenance functions gt gt Creating empty Results and Protocol Databases for archiving Archiving results records protocols stored curves and reader system and universal plate tests Deleting protocols from the current Protocol Database Deleting results records stored curves and reader system and universal plate tests from the current Results Database Utilities e 9 1 Incubation Control Incubate a plate now 9 2 e Utilities If the attached reader supports incubation the Incubation Control utility is available Use this utility to set the incubation setpoint temperature and duration then to turn on the reader s incubator and allow it to stabilize before performing a plate read e Note For the EL312I and EL340I readers the Incubation Control utility does not set or monitor the reader temperature This utility can however be used as a timer while the incubation temperature rises to and settles at a setpoint See step 6 for more information An alternative method for controlling incubation is to define incubation information in a
212. otocol Report for an absorbance assay showing Formula information 11 2 e Appendix A Sample Reports User s Guide to KCjunior Sample Report PROTOCOL TEMPLATE REPORT Protocol ID Sample Fluoro 1 Plate Read Time 11 18 99 4 08 26 PM Plate ID Sample Fluoro 1 Temperature n a General Information Raw Data Limits 0 to 99999 Discard Out of Range Values Blank Data Limits 0 to 99999 Discard Out of Range Values Decimal Points 3 Scientific Notation Raw Data Blanks Scientific Notation Transforms Concentrations Blank Method Full Read Method Information Chemistry Fluorescence Read Method Endpoint Filter Set 1 Excite 485 20 Emit 530 20 Bottom Sensitivity 75 Filter Set 2 Excite 485 20 Emit 530 20 Bottom Sensitivity 100 Filter Set 3 Excite 485 20 Emit 530 20 Bottom Sensitivity 125 Filter Set 4 Excite 485 20 Emit 530 20 Top Sensitivity 75 Filter Set 5 Excite 485 20 Emit 530 20 Top Sensitivity 100 Filter Set 6 Excite 485 20 Emit 530 20 Top Sensitivity 125 m p between filter set reads Geometry Reading Range A1 H12 Incubation ae Shaking n a ue a SOT 01 _ SKON RTOS E snr iron aro a srs war ID Dm cr le i Ae Da ile ie re r ie ee MPO PO MPO MP4 MPO POG MPO VIPOS aire MPIO MIE wie We Pe ioe vom Seed reol real rao rao rao ran rang e tit _cou MPO MPO P04 MPO MP06 MPO POS POS MPIO We 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 000 Sl MP0
213. ow x column matrix containing all defined Well IDs Click Show Formula to list the Transformation and or Plate Formulas gt The Raw Data Report contains the raw and or blanked optical density data or relative fluorescence luminescence data in one or more matrices corresponding with the plate geometry Click Raw Data Blanked Data or Both as appropriate Defining Protocols e 5 61 5 62 e Defining Protocols For MultiWavelength reads one matrix prints for each wavelength For Fluorescence and Luminescence reads one matrix prints for each filter set The Maximum Slope Report is available with kinetic protocols It reports the maximum slope calculated for every well in a matrix corresponding with the plate geometry For Fluorescence and Luminescence kinetic protocols with two filter sets defined two maximum slope values will print for each well one for each filter set The Formula Report contains the results of the Transformation Formula s and or Plate Formula in a matrix corresponding with the plate geometry Click Transformation Formula Plate Formula or Both as appropriate Click Show Formula to list the Transformation and or Plate Formulas The Column Report lists Sample IDs Well IDs well location up to three categories of measurement values and assigned cutoff labels if appropriate Information can be sorted by Sample ID or by location in the plate down or across If sorting is by Sample ID t
214. p down list gt Choose carefully it is important to select the correct model For example be sure to select ELx808I for an ELx808 with the incubation option gt Select PowerwaveX for any PowerWavexX model other than the PowerWave XS Do not select PowerWave340 or PowerWave340I for a PowerWavexX 340 instrument Setting Up the System e 4 3 If the EL301 is the current reader clicking Test Communications only verifies the parameter settings KCjunior will not attempt to communicate with the EL301 4 4 e Setting Up the System 10 11 12 13 14 15 16 17 Optional Click the Get Comm Defaults button to set the communication parameters to the default values associated with the selected Reader Type gt Ifthe current communication settings are acceptable jump ahead to step 16 Select a Com Port The Com Port is the computer s communication port that is used for data transmission between KCjunior and the reader gt Options include COM1 COM2 COM3 and COM4 Select a Baud Rate The Baud Rate is the transmission speed of data in bits per second between KCjunior and the reader gt Options include 1200 slow 2400 4800 9600 19200 and 38400 fast Valid settings vary among readers Set the Data Bits value to 8 Select a Parity setting Parity is a method of checking for errors during data transmission gt Options include None Odd and Even Set the Stop
215. pe wavelengths filter sets sensitivity kinetic settings number of plates microplate geometry incubation and shaking instructions and more Before Defining Read Method Parameters Because the availability of various Read Method parameters is reader dependent ensure that the desired Reader Type is selected under Setup Readers and also in the Current Reader menu before defining these parameters The reader does not need to be connected to the computer to define Read Method parameters Selecting a Read Method Type The Endpoint and Kinetic read methods are available for all readers supported by KCjunior For certain readers such as the PowerWave and the UQuant the MultiWavelength and Spectral Scan read methods are available For the FLx800 with its optional reagent dispenser the Dispense Read read method is available To select a Read Method Type click z to open the drop down list and select Endpoint Kinetic MultiWavelength Spectral Scan or Dispense Read e After a type is selected related definable parameters automatically become accessible For example if Kinetic is chosen data entry fields such as Kinetic Reads and Kinetic Read Interval becomes accessible e Sample Read Method dialogs are shown on the following pages 5 10 o Defining Protocols User s Guide to KCjunior User s Guide to KCjunior Read Method Dialogs for Absorbance Readers Protocol Definition General Information Read Method Temp
216. ppear follow the directions provided Help Topics KCiunior Hep RES Contents Index Find Ej KCjunior Samples Click a topic and then click Display Or clic Help System Contents re Introduction Getting Started Ne Sample Protocols amp Results Records KCjunior Samples NS Setting Up th hs ystem Defining Protocols Reading Plates Interpreting Results oO Printing Reports Utilities Troubleshooting NS Glossary Reviewing sample protocols and results records will help you to learn more about how to use KCjunior Please note that these are samples to be used for demonstration purposes only They have not been validated for use by Bio Tek Instruments Inc Descriptive text is provided for each sample to explain how the protocol parameters were defined Click here to continue Print Cancel Installing the Software e 2 15 Password Distribution and Software Registration 2 16 e Installing the Software You have 30 days from the date of installation to use KCjunior without a password After 30 days KCjunior will require you to enter a unique password in order to use the software This password can only be obtained from Bio Tek Instruments Inc If you are connected to the Internet you can get your password online and register your copy of KCjunior at the same time Registering your software with Bio Tek records your warranty and ensures that you will receive inf
217. ppropriate buttons gt KCjunior provides the option to perform a verification of the formula and check for syntax errors When the formula is fully assembled click the Verify button If the test is successful the message Formula is valid appears If the test is unsuccessful a message will appear describing the error s Edit the formula accordingly then click Verify again gt Click OK to close the Formula dialog and return to the Transformations Template The newly created formula will appear in the Transformation Formula field 2 Optional Click the Add to List button to save the new Add to List Transformation Formula 3 To apply the formula to all wells click the Apply to All Wells Apply To All wells button To apply the formula to specific wells use the mouse to Apply to Selection highlight one well or a block of wells to which the formula should be applied then click the Apply to Selection button 5 48 e Defining Protocols User s Guide to KCjunior KCjunior performs basic formula validation at this time For example if a formula references a well that is invalid for the current plate geometry a formula validation error message appears To perform a more thorough validation highlight the well containing the formula in question open the Formula dialog then click the Verify button 4 To access Copy Paste and Undo features click either the right mouse button or the Options button Working
218. protocol then click the Curve tab Select a different Stored Curve e Open the System Configuration Pathnames dialog Change the Results Database Path name to access a different database Too many standards for curve type Too many standards or unique dilutions titer assay were defined for the curve type e For example the 2P Logit Log and 4P curve types allow a maximum number of 24 standard groups 10 10 o Troubleshooting User s Guide to KCjunior Unable to generate curve The curve could not be generated Possible reasons include e No solution is available for the given set of points e If this is a titer assay there may be insufficient dilutions defined for the selected curve method e There are insufficient valid standards due to OUT masked suppressed or failed well conditions or other data reduction failures e The concentrations are all equal resulting in a slope of infinity Unable to initialize curve standards If this is a titer assay all replicates within a sample group are currently invalid e Invalid notations include gt OUT well is out of range gt well status error gt Strikethrough 2 765 well is masked User s Guide to KCjunior Troubleshooting e 10 11 10 12 e Troubleshooting User s Guide to KCjunior Appendix A Sample Reports This section contains sample KCjunior reports Reports are selected within a protocol under the Reports tab See Reports in Defining
219. ptions menu will appear Select V Mask Well to unmask the current well If the read is kinetic the well will only be unmasked in the current kinetic read Select Unmask All Reads to unmask the current well from all kinetic reads Interpreting Results e 7 37 7 38 e Interpreting Results Modifying Simulation Data If results were generated based on manually entered measurements the measurements can be modified from within the Raw Data results display To modify manually entered simulated data L 2 Click the Raw Data tab Place the cursor over the desired well and click the right mouse button once The Results Options menu will appear Select Modify Simulation Data The Simulation Well dialog will appear Simulation Well Dialog Well Location Read Number Read Value gt The Well Location represents the well s location in the microplate in row x column format gt Ifthe read is kinetic the Read Number represents the read during which the value for this well was measured gt The Read Value is the current value in the well Edit the Read Value then click OK to return to the Raw Data results display User s Guide to KCjunior Printing Reports Reports User s Guide to KCjunior After plate data has been acquired and data reduction is complete KCjunior can generate a user customizable set of reports Available reports include the Protocol Raw Data Maximum Slope Formula Column and Curve Repor
220. r retrieval and storage e The Results Database Pathname represents the location and name of the current results database gt Example pathname C KCjunior Database Results mdb e The results database pathname can be viewed or changed under Setup Configuration Pathnames Opening an Existing Results Record To open an existing results record begin by clicking the Open Results button in the KCjunior main screen selecting Results Open Results or by selecting a name from the Results drop down menu in the toolbar The Open Results dialog appears as shown below Open Results Dialog E AT III 11 29 2000 10 1510 AM B12 Assay 11 23 2000 10 49 31 AM Cancel SAMPLE Aflatoxin 6 23 1998 6 56 16 AM Hel SAMPLE Cytotoxicity 4 26 1998 10 51 06 AM Hb P SAMPLE Cytotoxicity II 6r2371996 1 10 36 PM SAMPLE HIF 6 25 1998 12 54 49 PM User s Guide to KCjunior Read Plate User s Guide to KCjunior e To open an existing results record highlight the desired Results record name in the list then double click the mouse button or click the Open button e To change the sort order click on the Results column header and or the Creation Date column header e To close this dialog without opening a record click Cancel or press the Esc key Generating a New Set of Plate Data Plate data can be retrieved from a microplate reader generated manually referred to as simulation data or read from a file To generate any n
221. r s Guide to KCjunior Appendix A Sample Reports e 11 11 Sample Report STANDARD CURVE REPORT Plate Read Time 06 23 98 08 56 16 AM Protocol ID Sample Protocol 7 Plate ID Sample Plate 7 Temperature n a Sample Curve Standards Suppressed Stds Standard Curve Interpolation Absorbance 300 400 200 ng I Interpolation Result 156 071 2 Parameter Logit Log y A D 1 x C B D A 1 3050 B 1 7104 C 131 4776 D 0 161 615 R Square 0 9987 04 23 99 01 17 04 PM Page of 1 Standard Curve Report 11 12 e Appendix A Sample Reports User s Guide to KCjunior SPECTRAL SCAN CURVE REPORT Protocol ID Sample Protocol 10 Plate Read Time 04 05 99 12 06 49 PM Plate ID Sample Plate 10 Temperature n a f 960 i i i i 4 560 640 720 800 880 Wavelength nm 04 23 99 01 32 09 PM Page of 1 Curve Report showing two overlaid spectral scan curves User s Guide to KCjunior Appendix A Sample Reports e 11 13 Sample Report TITER CURVE REPORT Plate Read Time 01 09 92 12 07 59 PM Protocol ID Sample Protocol 8 Plate ID Sample Plate 8 Temperature n a Sample Titer Curve Report toxicity 300 IFN_dilutn 4 Parameter y A D 1 x C B D 04 23 99 01 22 31 PM Page l of 1 Curve Report showing three overlaid titer curves 11 14 e Appendix A Sample Reports
222. r the value Click Curve Params to change any curve fitting parameters Click Edit Stds to suppress or restore one or more standards Note This feature is not available for titer or stored curves Alternatively click on a standard point in the graph to temporarily exclude it from curve calculations Click again to restore the standard Click Store the Curve to store a standard curve for use with other protocols Toggle the Display Legend setting to include exclude a legend in the graph Click OK to close the Standard Curve dialog Any changes to the standards will be reflected in the results calculations Click the right mouse button inside the graph to access advanced graphing options Interpreting Results e 7 21 7 22 e Interpreting Results Titer Curves If titer curves were generated clicking the Standard Curve button opens the Sample List dialog Sample List Dialog Es Select up to 8 Samples e Select up to 8 samples to be overlaid in one graph e Press the Shift key while using the mouse to select contiguous IDs ex SMPO1 04 e Press the Ctrl key while using the mouse to select non contiguous IDs ex SMP02 05 07 08 e Click the OK button to view the graph Standard Curve Dialog SAMPLE Titer Protocol Help Print Copy Titer Results Absorbance a SMPO1 SMPOS M Display Legend 7 8 9 10 11 12 13 14 15 416 Concentration 4 Parameter y 4 D 1 x C 8
223. ral Default Information KCjunior provides several configurable general system parameters including Startup Protocol Well Type Colors and Use Reader s Barcode Scanner for Plate ID To configure General Default Information 1 Select Setup Configuration Click the General tab The General dialog appears KCjunior Configuration x Well IDs Reporting Options General Pathnames Filter Wheel Cartridges m Startup Protocol C No Protocol Last Opened Protocol C Specific Protocol Well Type Colors Blanks Black v Samples Blue Standards Red v Controls Green v V Use reader s barcode scanner for Plate ID if supported by reader MV Remove first character of barcode typically defines the barcode standard i Cancel User s Guide to KCjunior Important If a database s location changes the pathname must be reconfigured User s Guide to KCjunior 2 Specify a Startup Protocol Instruct KCjunior to automatically open the most recently opened protocol a specific protocol or no protocol every time the software is started gt If Specific Protocol is selected choose a protocol from the drop down list 3 Change the Well Type Colors To differentiate between the four well types each can be set to a different color gt Available colors are Red Blue Green Yellow Magenta Light Blue Black Light Gray and Dark Gray 4 Ifthe attached reader has a barcod
224. re sending data to KCjunior e If plate data was read from an Omni System absorbance file the absorbance file may contain an invalid status 1 2 89 or 99 for that well indicating an error during plate processing or data reduction A memory allocation error has occurred KCjunior was unable to access system memory Suggestions e This error may occur if so many programs are open at once that system memory is overly consumed Close any unnecessary programs e Make sure that your computer meets the minimum system requirements listed on page 2 2 in Installing the Software BTI shared library could not be found The file BTI_ DLL DLL is missing from the Windows System directory Suggestions e Search your hard drive to see if the file has been moved If you find the file move it back to the Windows System directory e If you cannot find the file reinstall KCjunior Cutoff X could not be calculated A cutoff formula defined in the protocol could not be calculated Suggestions e Check the Cutoff dialog in the protocol to ensure that cutoffs are defined correctly e Ifa cutoff references a well ID ex PC this error may indicate that all replicates of the well ID are invalid 10 6 o Troubleshooting User s Guide to KCjunior User s Guide to KCjunior Cutoff X is not greater than the previous cutoff Two or more cutoffs are defined in the protocol and they are not in ascending order Suggestions e C
225. readsheet and paste it into a KCjunior plate Import plate data from an archive results database into the current results database Share plate data and results databases among multiple KCjunior users Read data from a KCJr for DOS plate file or from an Omni System absorbance file Export plate data to a text file or directly into a Microsoft Excel spreadsheet View store and access results Reading Plates e 6 1 Working With Plates and Results Upen Results Click on the Results column header to sort the list by name in ascending or descending order Or click on the Creation Date column header to sort by creation date and time 6 2 e Reading Plates This section describes accessing generating modifying storing and sharing plate data Where is Plate Data Stored Data for each plate is stored in a Results Record in the Results Database Each record is identified by a user assigned Results ID One results record stores all of the raw optical density fluorescence or luminescence values for a plate that was read once as with a single plate endpoint protocol or for a plate that was read multiple times as with a kinetic multi wavelength spectral scan or multi plate endpoint protocol Results Databases are typically stored on a computer s hard drive or on a network to which the computer is attached When opening or saving results records KCjunior automatically refers to the Results Database Pathname fo
226. ready open and then exports the data to the current Sheet User s Guide to KCjunior User s Guide to KCjunior Tip To avoid overwriting the contents of Sheet1 in Microsoft Excel choose another Sheet before performing the next data export Essential Exporting Information Certain numeric data is annotated by KCjunior when it is displayed Numeric data is exported however without these annotations Examples of annotations include Masked wells contain a strike through extrapolated concentrations are surrounded by and ambiguous concentrations are surrounded by exported exactly as it is displayed in KCjunior Data for all wells on the plate is exported even if the well is considered to be empty For all data options a well is empty if no ID is assigned to it For some data options such as Blanked Data a well is empty if its Raw Data value is currently masked The export utility is not available for the spectral scan read method Sample Output with Report Header and Data Descriptions Raw Data 405 0 000 0 1 42371 1 992 21 2 028 2 500 0 667 0 750 0 800 0 833 0 857 0 875 0 889 0 900 429 1 433 1 438 1 441 1 444 1 447 1 450 1 990 1 991 s99271 993 LeSO3i tl 2993 L993 22 0lL7 72 0097 2 0212 022 029 2 031 2 032 2 033 2 035 2 036 2 037 2 038 2 039 Sample Output with Empty Row Between Plates Kinetic 0 000 0 1 423 1 1 992 1 2 028 2 Kinetic 1 010 1 2 423 2 2298272 3 028 3 Re
227. rent dilution value To assign the next dilution change the value in the Dilution field then click one or more wells 4 To assign dilutions and let KCjunior increase the dilution values gt gt gt Enter the starting dilution value in the Dilution field The valid dilution range is from 1 0000e 99 to 9 9999e 99 Specify the Number of Replicates and Replicate Direction as explained in step 3 Select a Fill Count method of Delta or Factor Choose Delta to increase each dilution exactly by a defined value Choose Factor to increase dilutions by a defined factor value Enter a Delta Value or Factor Value as appropriate Specify a Map Direction of Down or Across This is the direction in which increasing dilutions should be mapped Highlight a block of contiguous wells to assign dilutions 5 To clear any mistakes gt gt gt gt User s Guide to KCjunior Set the Dilution value to 1 000 Set Replicates to 1 De select the Fill Count Highlight the well s to be reset to 1 000 Defining Protocols e 5 45 Click the Help button and then click the light bulb icon for tips on e Making a column wider or a row higher e Removing a transformation from a well e Highlighting all wells at once 5 46 e Defining Protocols Defining Transformations Transformations are user defined formulas applied to plate data for analysis or for data transformation before calculating concentratio
228. results after empty plate subtraction If Pathlength Correction is enabled the Corr option is presented for each wavelength This data set represents results after blank subtraction and pathlength correction e Pathlengths gt The Pathlengths option is available for MultiWavelength protocols with Pathlength Correction enabled e Max Slope gt The Max Slope option is available for Kinetic protocols to report the maximum slope calculated for every well gt For fluorescence luminescence Kinetic protocols with two filter sets defined Max Slope 1 and Max Slope 2 represent the maximum slopes calculated for every well for filter sets s 1 and 2 respectively e Transformations e Plate Transformations e Standard Conc Dilutions gt Standard concentrations or dilutions are included as they are defined in the protocol under the Curve tab e Calculated Concentrations e Cutoff Labels 5 64 e Defining Protocols User s Guide to KCjunior Reading Plates Overview The terms plate data and results refer to the set of measurements either retrieved from a microplate reader entered manually by the operator or read from an existing data file and the results of any calculations performed on those measurements Reading Plates describes how to use KCjunior to User s Guide to KCjunior Read a microplate with and retrieve plate data from the attached reader Simulate a plate read Copy data from a sp
229. ridges After selecting a cartridge clicking View displays the cartridge s contents Defining Protocols e 5 23 5 24 e Defining Protocols Filter Wheel Cartridge Dialog Name Ex Cartridge 1 Cartridge Type Excitation f Emission Position Wavelength Bandwidth 40 F Plug I Plug IV Plug i Plug gt Click Cancel to return to the Select Filter Wheel Cartridge dialog 5 Click OK to save the changes and return to the Read Method dialog The contents of the Excitation and Emission drop down selection boxes now match the selected cartridges Defining Filter Sets Each Filter Set specifies the Excitation filter Emission filter optics position sensitivity setting and sampling information to be applied during one plate read One to six filter sets can be defined for an Endpoint protocol One or two filter sets can be defined for Kinetic and Dispense Read protocols gt Note Dispense Read protocols have special filter position requirements See Filter Wheel Cartridges in Setting Up the System for more information There are many applications for the use of multiple filter sets including Comparison of results among various sensitivity settings with otherwise identical filter settings Assays that have more than one fluorophore per well Comparison of results between the top and bottom optics positions if the reader is equipped with dual optics Select two filter sets with identical settings
230. rmations 9 21 Deleting test results 9 22 Database version 4 10 Decimal places changing the number of 4 8 5 9 Delay before reading 5 32 Delay before sampling 5 28 13 2 e Index Delay between samples 5 28 Delete after archive 6 5 9 17 9 18 Delimiter choosing for data export 6 18 Delta OD 5 16 6 12 7 4 7 5 DILution function 5 51 Dilutions Defined 12 1 Defining 5 44 Valid dilution range 5 45 Dispense Read Read method type 5 14 5 21 Viewing results 7 14 Dispenser FLx800 Control panel 9 4 Filter position requirements 4 14 Initializing 9 5 Injector Position 9 7 Priming 9 6 Purging 9 6 Syringe setup 9 7 Display curve legend 4 9 Display settings 2 2 Displayed decimal places 5 9 Division in a formula 5 50 Dual reader support 4 1 4 2 5 3 E Editing standard outliers 7 25 Eject plate between filter sets 5 30 Emission filter 5 24 5 25 Emission filter wheel 4 11 5 23 EL301 microstrip reader 6 14 Empty plate MultiWavelength read 5 16 End of transmission character 4 4 Endpoint read 5 10 Defined 12 1 Multiple endpoint reads 5 10 5 20 Results 7 4 EOT character 4 4 Error code retrieving from reader 3 6 Errors and warnings A memory allocation error has occurred 10 7 Ambiguous concentrations 10 7 BTI shared library could not be found 10 7 Calculation could not be performed 10 5 Cutoff could not be calculated 10 7 Cutoff is not greater than previous 10 8 DIV 0 7 2 10 8 Error during auto sensitivity 10 8
231. rom every well on the plate e The values in the Standard wells are used to generate a standard curve e KCjunior recognizes individual Sample wells and sample groups if multiple replicates are defined The mean standard deviation and coefficient of variation are automatically calculated for each Sample group If defined in the protocol concentrations and cutoffs are calculated for each Sample replicate and group If dilutions are defined KCjunior generates a curve for each sample group e Up to five unique sets of Control wells can be defined see Customizing Well IDs on the next page This well type can be used to represent a typical control category e g positive control negative control or to fulfill a special requirement that the Blank Standard and Sample well types do not satisfy For example if an assay requires two unique Blank well identifiers define two sets of Control wells as follows Control 1 Blank H ELKI Control 2 Blank H2 BLK2 Note To perform blank subtraction for an assay using the Well IDs shown above a transformation formula resembling X BLK1 must be assigned to every well from which the BLK1 mean will be subtracted and X BLK2 must be assigned to every well from which the BLK2 mean will be subtracted See Working with Transformation Formulas in Defining Protocols for more information User s Guide to KCjunior Setting Up the System e 4 5 Customizing Well IDs Well I
232. rom a reader 1 Attach the reader to the computer and turn it on 2 Ensure that under Setup Reader the desired Reader Type is currently selected and that the communication parameters are set correctly 3 Click the Get Reader Wavelengths button or the Get Reader Filters button from the Read Method dialog KCjunior attempts to communicate with the reader If communication is successful the Wavelength Filter drop down menus are updated to match the reader s current settings Sending Reader Wavelengths For the PowerWave PowerWaveX and UQuant readers Just prior to plate reading if the protocol contains wavelengths that are not present in the reader s filter table KCjunior instructs the reader to calibrate the new wavelengths User s Guide to KCjunior Calibrating the Reader The Calibrate Reader button is available in the Read Method dialog for readers that support calibration When this button is clicked the reader runs a calibration routine and performs a system test Note For Spectral Scan reads there is a Calibrate Before First Read checkbox instead of a Calibrate Reader button See the next topic To calibrate the reader and perform a system test L Calibrate Reader 4 Ensure that under Setup Reader the desired Reader Type is currently selected and that the communication parameters are set correctly Ensure the reader is attached to the computer and turned on Ensure that the Wavelengths are
233. rotocols and Databases under Defining Protocols and Sharing Results Records and Databases under Reading Plates for more information 9 14 e Utilities User s Guide to KCjunior Creating Archive Databases Before protocols can be archived an Archive Protocol Database must be created Before results records stored curves system test results and universal plate test results can be archived an Archive Results Database must be created During the archive process selected items are copied from the current Results and Protocol Databases into the archive databases Look under Setup Configuration Pathnames to see the names and locations of the currently configured Results and Protocol Databases To create an archive database 1 Select Utilities Database Maintenance Create Archive Results or Protocol Database The Create Empty Results or Protocol Database dialog appears Create Empty Protocol Database E Save jni E Archives fag My Computer E 3 Floppy A E Ct J KCjunior Database File name Jarchive Protocols mdb Save as type Microsoft Access Databases mdb Cancel 2 Inthe Save in field select a location for the new database Enter an identifying File name such as ArchRs1lts mdb or ArchProt mdb 4 Click Save to create the database and return to the main screen Archiving Results Records and Protocols When results records and protocols are archived they are copied from the current Re
234. s Here you will assign dilutions for each sample group increasing by a factor of 2 1 The starting Dilution should be set to 1 000 by default 2 Set the Replicates Direction to 10 and Across respectively 3 Click Factor and set the Factor Value to 10 4 Set Map Direction to Down 5 Click and hold the cursor on well A3 Drag the cursor to D12 and release Set the starting Dilution back to 1 000 Click and hold the cursor on well E3 Drag the cursor to H12 and release The Dilutions Template should look like the one shown on the next page User s Guide to KCjunior Getting Started e 3 15 3 16 o Getting Started Protocol Definition Fa General Information Read Method Template Curve Cutoffs Reports C Well IDs Dilutions Transformations C Sample IDs SMPO02 SMPO3 SMPOS SMPO6 SMPOS 1 l1 1 ji Ja l SMPO2 SMPOS SMPO6 SMPOS Et 10 10 10 SMPO2 SMPOS SMPO6 SMPOS 100 100 100 100 100 100 100 SMP02 SMPOS SMP06 SMP03 1000 1000 1000 1000 SMP 12 SMP1i5S SMP16 SMP13 11 11 1 SMP 12 SMP1i5 SMP16 10 110 10 10 10 SMP12 SMP15 SMP16 100 101 100 100 SMP 12 SMP1i5 SMP16 Start Dilution r Replicates Direction m Fill Count fi fio Delta C Down Actoss M Factor fio Map Direction Help Down C Actoss Concentrations 8 Click the Transformations radio button Transformations Two typ
235. s ID in the Results Database User s Guide to KCjunior User s Guide to KCjunior Note The following sections discuss read method parameters that are specific to fluorescence and luminescence readers Skip ahead to page 5 31 for information on defining kinetic parameters enabling shaking or incubation validating read method parameters and more For general information on Read Method parameters or for instructions on selecting a Read Method type see page 5 10 Choosing the Chemistry Click the radio button associated with the desired chemistry Fluorescence or Luminescence e KCyjunior sets certain parameters in accordance with the chosen chemistry For example gt Measurements are reported as either RFU or RLU to represent Relative Fluorescence Units or Relative Luminescence Units respectively gt A warning message appears if the Excitation filter is not set to Plug in a Luminescence protocol a plug is recommended for Luminescence readings e For Luminescence protocols the instrument s lamp automatically turns off just prior to plate reading Refer to the instrument operator s manual for more information on the lamp Specifying Dispense Read Options If the current reader is the FLx800 and it is equipped with the reagent dispensing module you can choose to dispense reagent before or during sample measurement To define Dispense Read options 1 Ensure that the Read Method Type is set to Dispense
236. s that are running concurrently with KCjunior see System Requirements in Installing the Software for more information If the error persists with a fast kinetic read you may need to lengthen the kinetic interval Serial Read Error An error occurred when KCjunior attempted to communicate with the reader Suggestions e Make sure that the reader is on and ready to accept serial commands e Check to see if the correct serial cable is being used and that it is properly connected to the PC and the reader e Check that the communication settings under Setup Reader are correct for this reader The communication parameters can be tested from within the Setup Reader dialog Serial Write Error An error occurred when KCjunior attempted to communicate with the reader Suggestions e Make sure that the reader is on and ready to accept serial commands e Check that the communication settings under Setup Reader are correct for this reader The communication parameters can be tested from within the Setup Reader dialog User s Guide to KCjunior Troubleshooting e 10 3 Comments and Warnings reduction e The well references another well with a status of Well Read Error OUT Masked Divide by Zero Well Failure calculation within or involving the well attempted an invalid logarithmic function is syntactically invalid references an invalid location references an invalid Well ID e VV VV V F amp F VV VV V
237. saved User s Guide to KCjunior User s Guide to KCjunior Copying Protocols To copy the contents of an existing protocol to a new one with a different name begin by opening the protocol to be copied Once the protocol is open select Save As from the Protocol Menu KCjunior asks for a new protocol name Protocol Save As Dialog Ed Protocol Hame Cytotoxicity Il Cancel e Enter a new protocol name using up to 32 alphanumeric characters and spaces then click OK The new protocol is created saved and kept open for modification e Click Cancel to cancel this operation and return to the original protocol Sharing Protocols and Databases Since KCjunior stores protocols in a database there are several options for sharing protocols and protocol databases among users e A protocol database can be stored on a network for multi user availability gt Each user must configure the Protocol Database Pathname within his or her copy of KCjunior to point to the network database e A protocol database can be copied to a diskette or CD and exchanged between users gt If the database has grown too large to fit on a diskette create an empty protocol database and then copy only the desired protocols into the new database 1 Select Utilities Database Maintenance Create Archive Protocol Database The Create Empty Protocol Database dialog appears 2 Inthe Save in field select a location for the new database then enter an iden
238. ser You may transter the programs from one computer to another provided that the programs are used with only one computer at atime You may not electronically transter the programs from one computer to another You may not distribute copies of the programs or documentation without the prior written Do you accept all the terns of the preceding license agreement If sa click on the es push button If you select Wo Setup will close 10 Click Yes if you accept the terms Otherwise click No to discontinue the installation 2 6 e Installing the Software User s Guide to KCjunior e Ifyou click Yes the Registration dialog appears If you are running KCjunior Registration in trial mode and do not have a serial number enter Please enter the following Registration information any number in the Serial Number field Name Customer Mame Company Company Mame Seral fi 23456 Hack Cancel 11 Enter your name company name and the KCjunior serial number then click Next e The Registration Verification dialog appears Tou have provided the following registration information Hame Customer Hame Company Company Name Serial Number 123456 ls this registration information corect 12 Click Yes to confirm and continue or click No to make any changes User s Guide to KCjunior Installing the Software e 2 7 e The Choose Destination Location dialog appears Choose Destination Location Setup will
239. sorbance Readers Protocol Definition General Information Read Method Template Curve Cutoffs Reports Read Method Type Multiwavelength tad Help Wavelengths nm jo I Read Empty Plate Get Reader Wavelengths js0 V Read Empty Plate Validate Read Method E30 y MV Read Empty Plate Calibrate Reader a l Read Empty Plate m Pathlength Correction z e Head Empate M Use Pathlength Correction zE Bead Empi Plate MV Use Bio Cell Location at Plate Geometry ex1 2 x Read Al H12 Advanced Options Delay Before Read sec i 0 Monitor well E Monitor well Params Shake Method None hai Intensity Incubation Duration Temperature A ar ISGnUnUGUS Shake Cancel e During a MultiWavelength Read the plate is read at each defined wavelength up to 6 The optical density value measured at each wavelength is reported for each well Note The Pathlength Correction feature can be used with MultiWavelength reads Protocol Definition General Information Read Method Template Curve Cutoffs Reports Read Method Type SpectalScan First Scan Wavelength nm jo Last Scan Wavelength nm eo Scan Increment nm fio lneubatGn Duration lemperature Eontinuous Shake e During a Spectral Scan multiple readings are taken across a wavelength range on up to 8 wells A graph showing absorbance or transmission vs wavelength is reported for each well 5 12 e Def
240. ssing document then print the document Kinetic View Options i i ineti w Opti Calculation Options Hep C Raw Data Kinetic Curve Statistics Blanked Data Maximum Slope Sites e In this format each well initially displays a graph of the data points corresponding to the OD RFU or RLU readings taken over time e If blank subtraction was performed the kinetic curves are automatically based on the Blanked values e Click the Calculation Options button to adjust the kinetic read limits to alter the maximum slope calculation or to choose a different maximum slope unit of measure e To enlarge the view of the curve in an individual well place the cursor over the desired well and click the left mouse button The Kinetic Curve dialog appears 7 6 e Interpreting Results User s Guide to KCjunior Kinetic Curve Dialog Ed Plate SAMPLE Kinetic Plate gt Welk SMPOF G1 40 50 Seconds Slope 4200 00 milOD min R Square 0 9932 Help Print Copy x Axis Scale Seconds C Minutes C Hours The Kinetic Curve and its Maximum Slope and R Square values are displayed for the well gt Click OK to return to the Kinetic Curve results display Maximum Slope Kinetic read results are shown below in the Maximum Slope format Raw Data Template fe FR ee Fa Ea a Pa ae ee ee RS a 34 178 31 737 111 079 27 770 88 863 26 1
241. st readers is 2 C of the setpoint temperature Because of this KCjunior may provide the Place plate on carrier indication when the reader temperature is within 2 C of the set point temperature The incubator temperature will continue to rise and then stabilize at the set point temperature When the setpoint temperature is reached place the plate in the carrier then click OK The plate incubation will begin KCjunior will display the Time Remaining Incubation Dialog x m Temperature Control Incubator Set Point C Hep n e J Abort Incubation m Duration Indefinite Duration Seconds feo Time Remaining foo 54 Click the Abort Incubation button to stop the plate incubation When the incubation is complete remove the plate from the carrier 4 To turn the incubator on now and incubate a plate later gt gt Click the On radio button Enter the Set Point temperature The valid range for most readers is from 20 C or 4 C above room temperature whichever is greater to 50 C Utilities e 9 3 Turn the incubator off Set a timer Carrier Out In 5 gt Select Indefinite duration gt Click Set The Incubation dialog will close and the reader temperature will begin to increase or decrease appropriately To turn the incubator off gt Click the Off radio button gt Select Indefinite duration gt Click Set The Incubation dialog will close and the read
242. stall process may be incomplete if Windows was started by kipping past the password entry Select es to continue if a password was not required le it OK to continue with the install 8 If you were prompted for and entered a Windows password or if you were not prompted for this password click Yes e If you were prompted for a Windows password but did not enter one click No and go back to Step 1 e If you click Yes the following warning may appear Question A 2 The installation process will overnyite any pre existing E Cjunior files F The installation process will alzo replace any older OLL and OC files that are present and required by KCjunior le it OK to continue with the install 7 9 Click Yes to continue with the installation or No to cancel e If you click Yes the Software License Agreement appears Software License Agreement Please read the following license agreement Use the scroll bar to view the rest of this agreement BIO TEK INSTRUMENTS INC JLICENSING AGREEMENT YOU MUST CAREFULLY READ ALL THE TERMS AND CONDITIONS OF THIS JAGREEMENT PRIOR TO INSTALLATION INSTALLING THIS SOFTWARE INDICATES YOUR ACCEPTANCE OF THESE TERMS AND CONDITIONS If pou do not agree with these terms and conditions return the unopened package to Bio Tek Instruments Inc LICENSE Single Use fou have the non exclusive night to use the enclosed programs These programs can only be used by a single u
243. sults and Protocol Databases respectively into the archive databases To archive results records and protocols 1 Make sure that archive databases have been created 2 Select Utilities Database Maintenance Archive Results or Protocols User s Guide to KCjunior Utilities e 9 15 9 16 e Utilities The Archive Results or Protocol Database Pathname dialog appears Archive Protocol Database Pathname x Protocol Database Pathname C KCjunior D atabase Archives 4rchive Protocols mdb Browse Cancel Help Enter the drive directory and file name of the archive database or use the Browse button to locate this information Click OK The Archive Results or Archive Protocols dialog appears listing the contents of the current Results or Protocol Database Archive Protocols Dialog Ea Protocol Name Archive Protocols s Cancel Help V Delete after archive Select the results record s or protocol s to be archived gt If archiving results records click the Results and Creation Date column headers to sort the records alphabetically or by date gt Use the mouse to select the items s to be archived Press the Shift key while using the mouse to select contiguous items Press the Ctrl key while using the mouse to select non contiguous items gt Check Delete after archive to permanently delete the item s from the current database after being copied to the archive database Click the Archive Re
244. sults or Archive Protocol s button The selected items are copied to the archive database and then deleted from the current database 1f Delete after archive is checked gt Ifa duplicate Results Record or Protocol Name is detected KCyunior will not overwrite the results record or protocol User s Guide to KCjunior User s Guide to KCjunior Archiving Stored Curves When stored curves are archived they are copied from the current Results Database into the Archive Results Database To archive stored curves 1 2A Make sure that an Archive Results Database has been created Select Utilities Database Maintenance Archive Stored Curves The Archive Results Database Pathname dialog appears Archive Results Database Pathname x Results Database Pathname C KCjunior D atabase Achives Archive Results mdb Browse Cancel Help Enter the drive directory and file name of the archive database or use the Browse button to locate this information Click OK The Archive Stored Curves dialog appears listing the curves stored in the current Results Database Archive Stored Curves Dialog Ea Stored Curve Name Archive Curve s 4P Curve 1 AP Curve 2 Cancel Help Delete after archive Select the curve s to be archived gt Use the mouse to select the items s to be archived Press the Shift key while using the mouse to select contiguous items Press the Ctrl key while using the mouse to select non cont
245. tatus and temperature e Click z to open a list of existing results records e Click to read a new plate e Click to save the current results record e Click to close the current results record Use the icons on the right side of the toolbar when working with protocols B12 Assay fC mle a AT lll B12 Assay Cell Count Color Comb B0 e A protocol contains instructions for operating the attached reader and for performing data reduction on the raw data retrieved from the reader e Click z to open a list of existing protocols e Click 2 to modify the open protocol e Click to create a new protocol e Click to save the current protocol e Click to close the current protocol User s Guide to KCjunior User s Guide to KCjunior Results Menu The Results Menu contains functions for acquiring and processing microplate data sie Protocols Utilities Set Read New Plate Ctrl F Open Results Ctrl 0 Close Results Plate Information Save Results Lhrl 5 Save Az Inport Results Esport Data Print Results Ctrl F Print Preview Print Setup E xit Read New Plate Open Results Close Results Plate Information Save Results Save As Import Results Export Data Print Results Print Preview Read a new plate Open an existing results record Close the open results record If the record has not yet been saved KCjunior provides the option to save before clos
246. teade Dispenser COnuol PAMe lh iat cutaceucciacste tarsi teen aN Initializing the Dispenser s sceceeeeeeeeeeeeees Priming the Fluid Lanes enoei sa Purging the Fluid Lines esei Setting the Injector Position eeeenessssssseeee0eeeee SYDSES SCLUP naaa A DAINOS S eaa a Reader System Testori on a Universal Plate TeS bteancogancnirepan Database Maintenance ccccccccsssssssesseeeeeeceeeceeeeeeeeaeeeeeeeees A Note on Backup Procedures ccccceeeeeeeeeees Creating Archive Databases ccccccceeeeeeeeeeees Archiving Results Records and Protocols Archiving Stored Curves ssseesseeeeeeeeeeeeeeeeaes Archiving Test Results cccssssssssssseeeeeeeeeeeeeeeaes Deleting Results Records and Protocols Deleting Stored CURVES wassnarcea sites car liesiatanteaceweceinces Deleting Stored Transformations ccccceceeeee Deleting Test Results mdai Troubleshooting VSI VIS Wase T E E eon aaa oss Reade C EITOL eRe eae RA DRE a a AVON tA OUR NO yA Communication Errors enaa a a i Comments and Warina Sareri cadvauees Appendix A Sample Reports Glossary Index iv e Contents User s Guide to KCjunior Introduction KCjunior User s Guide to KCjunior KCjunior interacts with and performs data analysis for select microplate readers With KCjunior you can Configure the software to communicate with two Bio Tek readers and switch e
247. ted item s are permanently removed from the current Results Database User s Guide to KCjunior User s Guide to KCjunior Deleting Stored Transformations If certain stored transformations are no longer needed they can be deleted from the current Protocol Database To delete stored transformations 1 2A Close any open protocols Select Utilities Database Maintenance Delete Stored Transformations The Delete Stored Transformations dialog appears listing all formulas stored in the current Protocol Database Delete Stored Transformations Dialog Eg Delete Formula lt BLK1 CTRL1 BLK1 100 lt BLK2 CTRL2 BLK2 100 Help CTRL1 100 Select the formula s to be deleted gt Use the mouse to select the items s to be deleted Press the Shift key while using the mouse to select contiguous formulas Press the Ctrl key while using the mouse to select non contiguous formulas Click the Delete Formula s button The selected item s are permanently removed from the current Protocol Database Utilities e 9 21 9 22 e Utilities Deleting Test Results After System Test and Universal Plate Test results have been safely archived or if certain test results are no longer needed they can be deleted from the current Results Database To delete stored System Test or Universal Plate Test results 1 2 Close any open plates Select Utilities Database Maintenance Delete System Test Results or Delete Univ
248. the file name already exists KCjunior provides the option to overwrite the entire contents of the file or to append the new data to the end of the file Note If exporting data for a multiple plate endpoint read the Export Endpoint Plates dialog appears Export Endpoint Plates Dialog Number of Plates Cancel First Plate to Export Last Plate to Export Specify the First and Last plates to export then click OK to continue Export format All selected data sets are exported for the first plate then for the second plate and so on through the last plate User s Guide to KCjunior Select Results Export Data to open the Export Data dialog After each export KCjunior retains the list of Selections up to 15 for use with the next export This is particularly useful if exporting the same set s of plate data for multiple plates User s Guide to KCjunior Exporting Data Directly to Microsoft Excel To export data to Microsoft Excel I From within the Export Data dialog choose the Export Destination option Directly to Microsoft Excel If exporting to an existing Excel spreadsheet gt gt gt gt Click Start Excel now to launch Excel immediately Open the spreadsheet Review the spreadsheet to determine what data is expected and where it should be stored Important If the spreadsheet contains formulas or other important information take the following into consideration to avoid overwritin
249. then double click to insert Defining Protocols e 5 59 gt Mathematical values and symbols can be entered manually or selected by clicking the appropriate buttons gt Location indicators can be entered manually or selected by clicking the appropriate buttons gt When the formula is fully assembled click the Verify button to check for syntax errors If the test is successful the message Formula is valid appears If the test 1s unsuccessful a descriptive message appears Edit the formula accordingly and click Verify again gt Click OK to close the Formula dialog and return to the Cutoff dialog The newly created formula will appear in the appropriate Cutoff field 5 Specify the text to be associated with each cutoff zone in the Label fields These labels will be assigned to wells after evaluation gt Each label can contain up to five alphanumeric characters gt The number of labels must exceed the number of cutoff values or formulas by one gt Inthe following example labels will be applied to wells as follows Neg if the value in the well is less than 10 0 Eqv if the value in the well is greater than or equal to 10 0 and less than 20 0 Pos if the value in the well is greater than or equal to 20 0 Cutoff 5 60 e Defining Protocols User s Guide to KCjunior Appendix A at the end of the User s Guide contains sample reports Most reports require that well identifiers be assigned in the
250. this mode in the future Just before the plate read KCjunior checks to see if the attached reader supports the Read Mode currently selected If the reader does not support the current Read Mode a different one must be selected Defining Protocols e 5 35 Validate Read Method Get Reader Wavelengths Get Reader Filters 5 36 o Defining Protocols Validating Read Method Parameters The Validate Read Method button is available in the Read Method dialog for some readers When this button is clicked KCjunior communicates with the reader to verify the defined Kinetic Interval and also verifies that all required fields contain valid entries To validate the read method parameters 1 Ensure that under Setup Reader the desired Reader Type is currently selected and that the communication parameters are set correctly 2 Ensure the reader is attached to the computer and turned on Define the read method parameters 4 Click the Validate Read Method button KCjunior attempts to communicate with the reader If communication is successful the parameters are validated and the results are displayed If a change is required KCjunior provides the necessary instructions Retrieving Reader Wavelengths Filters KCjunior provides the capability to retrieve wavelength filter information from the attached reader and store that information in the drop down Wavelength or Filter selection menus To retrieve Wavelength Filter information f
251. tic to the upper and lower standard values The four parameters are left asymptote right asymptote slope and slope at the inflection point Point to Point Follows each standard point with no averaging of the values to smooth the curve Cubic Spline A piecewise polynomial approximation consisting of joining a set of data points by a series of straight lines which is then smoothed by using a cubic fit Enter a name for the graph in the Graph Report Name field using up to 16 alphanumeric characters gt This name appears on Standard Curve reports Select a Y Axis Scale of Linear or Log gt For most curve methods if a logarithmic scale is selected then logarithmic numbers are used for both the calculation of the curve and the plotting of the curve For the 2 Parameter and 4 Parameter curve methods however the logarithmic scale is applied only to the plotting of the curve The curve is calculated using linear numbers User s Guide to KCjunior User s Guide to KCjunior 10 11 12 Specify a Y Axis Label using up to 32 alphanumeric characters This label appears on displayed and printed reports gt Common labels are Optical Density RFU and RLU Select an X Axis Scale of Linear or Log gt For most curve methods if a logarithmic scale is selected then logarithmic numbers are used for both the calculation of the curve and the plotting of the curve For the 2 Parameter and 4 P
252. tifying File name 3 Click Save to create the database and return to the main screen 4 Select Utilities Database Maintenance Archive Protocols The Archive Protocol Database Pathname dialog appears Defining Protocols e 5 5 5 6 e Defining Protocols 5 Enter the drive directory and file name for the newly created empty database 6 Click OK The Archive Protocols dialog appears listing the contents of the current Protocol Database 7 Select the protocol s to be copied Do NOT check Delete after archive 8 When all items have been selected click the Archive Protocol s button The selected items are copied to the new database Protocols can be imported from one database into another See Importing Protocols below for more information Suggestions gt Protocols can be imported from a master protocol database stored on a network gt Protocols can be imported from a protocol database stored on a diskette or on a CD Importing Protocols Protocols from one Protocol Database can be imported into another The Protocol Database that will receive the imported protocols is the one defined under Setup Configuration Pathnames To import protocols from another database L Ai If a protocol is open close it Select Protocol lmport Protocols The Import Protocol Database Pathname dialog appears Import Protocol Database Pathname Ea Protocol Database Pathname C KCjunior D atabase M
253. ting data 6 19 Geometry 5 32 ID 4 10 5 20 7 35 6 8 6 9 9 10 Importing 6 5 Opening a results record 6 2 Partial plate reading 5 32 Plate status 7 35 Reading 6 15 Saving 6 4 Shaking 5 34 Plate formula 5 46 5 49 Plate geometry 5 32 Plate ID 4 10 5 20 6 9 6 11 6 18 Multi plate read 6 9 6 10 6 11 Plate information 7 34 Plate mode reading 5 21 Point to Point curve 5 56 PowerWaveX reader models 2 14 4 3 User s Guide to KCjunior Primary wavelength 5 16 Printing reports 5 61 8 1 Print preview 8 1 8 2 Print results 8 2 Print setup 8 3 Product information 1 3 Protocol Blank limits 5 9 Copying 5 5 Defined 3 9 Deleting 9 19 Description 5 8 Importing 5 6 Name 5 8 Raw data limits 5 8 Read Method Type 3 11 5 10 Report Options 5 8 Saving and closing 5 4 Sharing protocols among users 5 5 Protocol database Archive protocol database 9 15 Creating an empty database 5 5 Importing protocols 5 6 Pathnames 4 10 PROTOCOL MDB 4 10 Sharing protocols and databases 5 5 Version 4 12 Q Quadratic curve 5 56 R Rapid read mode 5 35 Raw Data 7 4 Printing 8 1 Viewing results 7 1 Raw data limits setting 5 8 Raw Data report Selecting 5 61 Printing 8 1 Read from file 6 3 6 13 Read full plate 5 32 Read Method Auto sensitivity adjustment 5 28 Calibrate before first read 5 37 Calibrate Reader 5 37 6 8 Chemistry 5 21 Delay Before Read 5 32 Fastest kinetic interval 5 31 Filter sets 5 25 Get Reader Wavelengths 5 16 5
254. tion Results Transformation Formulas are applied according to a certain structure See Essential Information in Getting Started for more information User s Guide to KCjunior Transformation Results are displayed if at least one Transformation Formula was applied to one or more wells on the plate Raw Data Transformation Results Concentrations Template Comments Warnings 0 667 0 800 0 833 i 0 889 0 900 1 433 1 441 1 444 1 990 1 991 1 993 1 993 1 993 3 2 021 2 022 2 031 1 683 2 035 1 688 1 839 1 769 1 400 2 230 i 2 231 1 402 1 574 2 174 2 235 i 1 576 1 426 1 757 1 428 1 548 2 833 3 235 2 843 j 2 846 2 848 2 852 2 853 Statistics Standard Curve Each well contains the result of its Transformation Formula if one was applied Otherwise the well contains its Raw Blanked or Corrected value For Kinetic reads Transformation Formulas are applied to the Maximum Slope For MultiWavelength reads Transformation Formulas are applied to the Raw or Blanked Data for the first defined wavelength unless the WV function is used to specify a different wavelength For multiple Filter Set reads Transformation Formulas are applied to the Raw or Blanked Data for the first defined filter set unless the FS function is used to specify a different filter set See Defining Transformations Formula Syntax in Defining Protocols for
255. trations based on the curve generated using the present standards or on a previously stored curve Generate a curve using the sample dilutions as standards Extend the abscissa range of a standard curve fitting routine beyond the limits of the standard data points generated by the assay extrapolation Edit standard outliers and recalculate the curve Run a Universal Plate Test on most readers and then view print and save the test results Run a System Test on most readers and then view print and save the test results Test communication between KCjunior and the reader before attempting to read a plate Set default communication parameters appropriate for the attached reader Control plate incubation reader dependent User s Guide to KCjunior Technical Support If KCjunior fails to function successfully try to solve the problem s with the assistance of the Reader Run Time Errors Comments and Warnings and Troubleshooting sections of the User s Guide or the Help system If the software still does not function successfully 1 Select Help About KCjunior from the KCjunior main screen A dialog resembling the following appears About KCjunror E KC E Ciunior Installation f BTlLab32 dll Reader Control Version KOIrwin exe Results Database Version Protocol Database Versior This product ts licensed to A Customer Bio Tek Instruments Ine 123456 Copyright Bio Tek Instruments Ine 199
256. trough is in place on the carrier see the FLx800 operator s manual for placement instructions In KCjunior select Utilities Dispenser Control Panel Click Purge Dispenser Enter the desired Purge Volume from 5 to 5000 ul go See ee ee Click Start Purge to begin the purge 9 6 e Utilities User s Guide to KCjunior Setting the Injector Position The FLx800 s fluid injector head can be positioned either next to the top probe or directly above the bottom probe Refer to the FLx800 operator s manual for details If the fluid injector head is moved the reader software must be reconfigured accordingly this can only be done through KCjunior To configure the injector position setting 1 In KCjunior select Utilities Dispenser Control Panel 2 Click Set Injector Position 3 Click the radio button associated with the Injector Position next to top probe or above bottom probe 4 Click Set Position The message Injector position setting successfully completed should appear 5 Click OK to return to the Dispenser Control Panel The Injector Position field will display the new position Syringe Setup KCjunior provides access to special Syringe Setup functions for maintenance and calibration purposes e Important Do not change the syringe position or calibrate the dispenser unless instructed to do so as part of installation upgrade or maintenance Moving the Syringe to its Maintenance Position If the syrin
257. ts as well as the configurable Matrix Report Reporting options are selected and customized within a protocol under the Report tab See Defining Protocol Parameters Reports in Defining Protocols for more information Note A protocol must be open to print most reports If no protocol is open however a Raw Data Report containing just the raw plate data can be printed Print Preview Before actually printing reports select Results Print Preview to see what they will look like e For multiple plate endpoint reads the Print Endpoint Plates dialog appears Print Endpoint Plates Dialog Humber of Plates Cancel First Plate to Print Last Plate to Print gt The Number of Plates field contains the total number of plates that were read gt Adjust the First Plate to Print and Last Plate to Print values to preview a subset of the total number of plates gt Click OK to continue with the preview Printing Reports e 8 1 8 2 e Printing Reports e The Print Preview dialog can display one or two pages at a time gt Click HEHE PERE to view the next page of the report gt Click Pees PELE to view the previous page gt Click Zoran or to change the view gt Click Me to access the Print dialog to select a printer and related options gt Click es to close the print preview dialog and return to the KCjunior main screen Print Results Select Results Print Results to print the reports e If
258. ueimsnen a asaveadanes 6 7 Reading Flate Without a Protocol essien Ghee teinel ede 6 7 Reading a Flate Withsa PrOtocol uss satcsnacearucinaelaiteadeedbipaniutentcmopinaaiatendendioiiideace 6 10 Simulating a Plate Read osi E E teases ea caiataataGes 6 12 Reading Plate Data Tom a Pilesa Wied E a a 6 13 Collecting Data from an EL301 Microstrip Reader cccccseseeeeseeeeeeeeeeeeeeeeeeaes 6 14 Reader Runtime Ero arai ean A catanuaseatwat 6 16 Come MS Warn Seaia AE E 6 16 selecting a Filter Wheel Carrido a A 6 17 Eponina Dala aoa a 6 18 Vie WU RCS US eeen E N NN 6 24 Interpreting Results 7 1 ONEIN E Wene TOT NA O OAN 7 1 W ELE INOLALIONS oaei E A E E aude 7 2 IRA WDA E EEEE EE EE E ounce pe otee TEE EAEE cations ec P TATE naan A E TA ATTE 7 4 Raw Endpoint Read Data ohen ega ea a a a E aaia 7 4 Raw and Blanked Kinetic Read Data ccccccccccccccceesssseeeeeeeeeeeceeeeeeeeeeeaaaaaeeneees 7 5 Raw and Blanked Special DACA xiii a a a ded Mawes EN 7 9 Raw MultiWavelength Read Data cccccccccccccccccceeeeseseeeeeeeeceeeeeeeeeeeaaaaeeeeees 7 13 Raw Dispense y Read Datarea a A 7 14 Binke DIE O 7 16 Blanked Endpoint Read Datirerssare oiana A 7 16 Blanked MultiWavelength Data cccccccccccccecceceeceeeeeeesesseeeeeceeeeeeeeeeeaaaaeesesees 7 17 Patiale moth C On ec WO toss cass a ecxeca tase ahead ateweataeiin a hdc woes uss eeadieeea das 7 17 TV PANSTOLMANOM Resul esni a a ve tealaaaiorsaas dee tdea s
259. ula Results Concentrations and Cutoffs User s Guide to KCjunior Quick Start User s Guide to KCjunior If Transformation Formulas are applied Transformation Results become the basis for calculating Concentrations and Cutoffs If Concentrations are calculated they become the basis for Cutoffs evaluation Notes gt For Kinetic protocols the Maximum Slope is the basis for calculating transformations for curve calculation if there are no transformations and for cutoff evaluation if there are no transformations or concentrations gt For Dispense Read protocols FLx800 Raw Data is the only result KCjunior does not support blank well subtraction pathlength correction transformations curve fitting or cutoffs for this read method type When using KCjunior you will typically follow some version of the steps shown below oY a ee oe NY E Start KCjunior Connect a microplate reader to the computer Select Setup Reader to define reader setup parameters Select Setup Configuration to set default system parameters Select Protocol New Protocol to create a new protocol or Protocol Open Protocol to open an existing one gt Define read method parameters including read method type wavelengths and plate geometry gt Create a Well ID template gt Define Curve parameters and Cutoff criteria if required gt Define Reporting options Select Plate Read Plate to read a microplate Review results
260. ve Dialog Stored Curve Name Cytotosicity Il Cancel Enter a unique name and click OK to store the curve and return to the Standard Curve dialog Click OK to close the Standard Curve dialog and return to the Concentrations results display User s Guide to KCjunior Labels Labels are displayed if cutoffs are defined in the protocol Raw Data Labels Template Cutoff Values Help Statistics e ach well contains the Label assigned to it after cutoff evaluation e For Endpoint reads cutoff evalution is based on the OD RFU or RLU values e For Kinetic reads evaluation is based on the Maximum Slope values e For MultiWavelength reads evaluation is based on the measurements at the first defined wavelength unless Transformation Formulas utilizing the WV function are applied to specify a different wavelength e For multiple Filter Set reads evaluation is based on the measurements at the first defined filter set unless Transformation Formulas utilizing the FS function are applied to specify a different filter set See Defining Transformations Formula Syntax in Defining Protocols for more information on the WV and FS functions e Click the Cutoff Values button to see the cutoff criteria the calculated cutoff value s and the labels assigned to each cutoff zone User s Guide to KCjunior Interpreting Results e 7 27 Template 7 28 e Interpreting Results Well identificat
261. well monitoring continuous shaking for kinetic reads reader calibration for spectral scan reads filter wheel cartridge selection the DILution function and some new reporting options Updated Reading Plates to discuss the new Results ID using the reader s barcode scanner to retrieve the Plate ID and selecting a filter wheel cartridge at run time Updated Interpreting Results to include selection of the Maximum Slope Unit of Measure Added new sample reports to Appendix A Updated Installing the Software with the correct website address for software registration Updated Setting Up the System to include filter position requirements for Dispense Read protocols Updated Defining Protocols to introduce new Read Mode selections as well as the new Dispense Read method type for the FLx800 Updated Interpreting Results to describe Dispense Read results Updated Utilities to describe the new Dispenser Control Panel feature for the FLx800 Contents Introduction 1 1 KE OTOL anann E S S l 1 Technical Suppor ecr e e E E a e E a a 1 3 Visiting Bio lek s WEbsifenenianacrae anra aae a aa a a ar 1 4 Installing the Software 2 1 Read Mel siicesstaveiotea oan a E RN 2 1 Sys em Regu re Men S meaa a E a a EN 2 2 Removing an Earlier Version of KCJUMIOL cccccccccccccssssssssssessseeececceeeeeeeeeeaasasssesseeeeeeeees 2 3 TOTEMOVE K CJUNO eio n o a OE EOE 2 3 SOltw are Me O aa e a a A me coacecdoscseceesossecaees
262. word This helps to ensure a complete installation If you are not prompted with the Windows password entry disregard this note Close any unnecessary Windows programs Place Disk 1 in a 3 5 diskette drive or the CD in the CD rom drive Click the Windows Start button and select Run When the Run dialog box appears type lt drive gt setup exe where lt drive gt represents the letter corresponding to the diskette drive or CD rom drive e Alternatively use the Browse function to access the drive then click on setup exe Click OK After a brief pause the Welcome dialog appears eal This program will install KEjunior on your computer It is strongly recommended that you exit all Windows programs before running this Setup program Click Cancel to quit Setup and then close any programs you have running Click Next to continue with the Setup program WARNING This program i protected by copyright law and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and violators Will be prosecuted to the maximum extent possible under law Cancel If you accept these terms click Next Otherwise click Cancel to discontinue the software installation Installing the Software e 2 5 e Ifyou click Next the following question appears For systems that require a password when starting Windows The installation and Unin
263. ys a standard well contains a grayed value this indicates that the standard has been suppressed e To restore the standard click Standard Curve click Edit Stds then change the Status of the desired well illegal log function attempted on a standard This error typically results during curve generation if the Y Scale value is set to Log and the OD RFU or RLU value for a standard or dilution well is 0 000 e For titer curves the warning will report the Sample ID s experiencing the error Insufficient valid standards for this curve type To generate a successful curve the selected curve method requires more standards than are currently valid on the plate Suggestions e Check the Raw Data values for the standards If one or more standards is reported as OUT this means that the OD RFU or RLU value fell outside the minimum maximum data range defined in the protocol and is excluded from further calculations e If you just edited one or more standard outliers there may now be too few remaining valid standards e The minimum number of required valid standards for each curve method are Linear and Point to Point 2 Quadratic and Cubic Spline 3 Cubic 2 Parameter and 4 Parameter 4 No valid blanks in sector X All blank wells in one or more sectors in the plate are invalid Suggestion e Ifall blank wells in a sector are reported as OUT the Raw Data values for these wells exceeded the minimum maximum dat

KCjunior Users Guide - Bio-Tek

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