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Gen5™ & Gen5 Secure User's Guide
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1. A protocol is a recipe or set of instructions designed to capture transform and report and or export data Protocols are created and saved as standalone files They function as a template an unlimited number of experiments can be based on one A protocol consists of reading requirements like detection method and wavelength and reading related actions like shaking and incubation Procedure plate layout data reduction and data viewing reporting and exporting definitions A protocol can be used repeatedly as is or modified within experiments By itself a protocol does not produce results Protocols do not have plates associated with them prt is the protocol s filename extension A copy of the protocol is saved within an experiment or as a standalone prt file Since protocols do not have plates they cannot generate data outside of an experiment The Gen5 Secure level of software maintains an audit trail of all activity and changes related to a protocol All other Gen5 software levels do not support this feature An experiment has a copy of the protocol and at least one plate It executes the instructions provided by the protocol to produce results While an experiment is created using an existing protocol the experiment s copy of the protocol can be modified within the experiment Running an experiment is the only way to process a protocol Gen5 s Quick Read function may at first appear to skip
2. DS1 D S2 to subtract the reference wavelength 630 measurements from the test 410 measurements Retain the default Use single formula for entire plate Click OK to save and close the Transformation e Click Curve Analysis In the Data In tab use the drop down list to select Y D ata Dual Wavelength and click OK For this simple protocol the remaining default settings are acceptable More options are available like customizing the names of data sets plotting interpolations in the generated curve and so on See Plotting a Curve in the Data Reduction Options chapter e Click OK to closethe Data Reduction dialog Set the Report parameters and D ata Views as desired For instructions see Viewing Results Save the protocol select File Save and nameit D ualWavel for this example Run the protocol Now you re ready to run the DualWavel protocol in an experiment l Select File New Experiment By default Gen5 highlights the DualWavel protocol in the dialog making selection quick and easy If the reader is all set up you re ready to go Click Read and follow the online prompts 64 Chapter 4 Assay Examples Basic Spectrum Analysis Numerous applications can profit by a preliminary spectral screening H ere are instructions for setting up a basic spectrum protocol in Gen5 Q It may be easier to follow these instructions if you have already watched the Gen5 Basic series of online tutorials select H elp gt Tut
3. directory select the Samples folder e All other levels of Gen5 Select File Open Protocol and browse to C Program Files BioTek Gen5 Samples e Gen5 sWelcome screen also offers the option to open a Sample File Important The sample protocols must be considered as examples provided for demonstration and guidance purposes If you plan to use these protocols or similar ones in a real application it is your responsibility to validate the protocol parameters including the report and export content if applicable before using them Notes Your system administrator can change the path and filenames described above If you cannot find the Samples folder contact your system administrator Also note your reader may not support all of the sample protocols provided Review the descriptions in the Samples Protocol Listing to see if your reader is compatible with the defined steps Sample Protocols and Experiments Guide Y ou can review a complete description of samples in the Sample Protocols and Experiments Guide PDF shipped with Gen5 dick the Windowse Start select All Programs gt G en5 gt Sample Protocols and Experiments G uide 4 Gen5 installs a copy of the Sample Protocols and Experiments Guide in the Samples folder of the main Gen5 directory By default this is C Program Files BioTek Gen5 Samples Sample Protocols and Experiments 53 Y ou can also find a summary listing and brief description of the sample protocols in Ge
4. 260 nm is 13 ODs for a 1 cm pathlength this can be recalculated to mean 1 0 OD has a concentration of 32 5 ug ml Save the protocol Now you re ready to define your reporting requirements and run the protocol in an experiment 62 Chapter 4 Assay Examples Dual Wavelength Absorbance Endpoint Here are step by step instructions for setting up a dual wavelength absorbance read with known concentrations of standards against which a linear regression curve is plotted Create the protocol l 2 3 5 Select File gt New Protocol Er ir Double dick Procedure in the menu tree e Click Read to set the reading parameters Keep the default settings for Detection M ethod and Read Type Absorbance Endpoint e For Wavelengths click the button for 2 and usethe drop down list to select or enter the test and reference wavelengths 410 and 630 for this example e Click OK twice to close the Read Step and then the Procedure dialogs ues Double click Plate Layout to define the location of standards samples and blanks on the microplate For this example the standards are placed in the center of the plate modify the instructions to match the distribution of samples and standards on your plate e Setthe Well Settings Type to Standard and click the 3 dot button next to the Conc field to enter the expected concentrations For this example leave O in the STD1 cell at thetop of thetable Select Incr with a tick mark
5. databases see Maintaining Files for instructions on reducing the database size Consider using Gen5 s automatic Save feature to create a new date stamped folder for storing experiment records This is an especially good practice for large labs with multiple users who run hundreds of plates per day Gen5 will keep all that data organized by date Definethis kind of file management setting in the Default Protocol so it will apply to all newly created protocols Turn off the Multi Read Calculation option to improve Gen5 s performance Calculation results will be the same but your PC s resources will not be diverted for performing interim calculations Best Practices 49 Time Savers Partial Plate for assays using strips or partially filled plates especially if the read steps are long or complicated you can save time by telling the reader exactly which wells or portion of the plate to read D efault Protocol all newly created protocols and Quick Reads are based on the Default Protocol If some protocol elements like plate layout runtime prompts report headers and footers etc are largely the same for most of your projects you ll save significant time by defining these elements before creating the next protocol experiment Print Preview save time and paper by viewing reports on screen before sending them to the printer 50 Chapter 3 Essential Concepts Chapter 4 Assay Examples This section contains step by step instructi
6. 1S SPL18 1 STDS STES SPL3 SPL3 SPL3 SFL11 SFL11 SPL11 SPLI9 SFL18 SPL18 0 0 51049 51049 SPLA SPL4 SPLA SPL12 SPL12 SPL12 SPL2O SPL20 SPL20 30 30 30 STDS STDS STES SPL5 SPLA amp SPLA amp SPL13 SPL13 SPL13 SPL21 SPL21 SPL21 a a 4 STOG ST DG 7 DG SP LEG SPL6 SPL6 SFL14 SPL14 SFL14 SPL22 FLZZ DIESES fae Ae e EG ol a D STD STD STD SPL SPL SPL SPL15 SPL15 SPL15 SPL23 DEAE T E d d SPLZ23 The critical factor is using the Well IDs not their location on the plate We did not need to customize the Well IDs for this example We simply selected the Type defined the known concentration of the standards and assigned them to the plate Blank DI water e EEE Standard Known concentrations CTL1 Assay Control Known Control SPL Sample Unknown samples Find specific instructions in the Preparing Plates chapter 3 Defining the Data Reduction Steps Now that we ve defined the reading parameters and plate layout we can define the data reduction steps blank well subtraction standard curve and expressing samples as a percentage of the control Gen5 creates the blank subtraction step for you automatically l Select Protocol Data Reduction Notice that one Transformation named Blank nnn where nnn is the wavelength has already been created We ll use the results of this calculation to plot the standard curve Click Curve Analysis Quantitative ELISA Exa
7. Gen5 amp Gen5 Secure User s Guide Microplate Data Collection amp Analysis Software BioTek I nstruments I nc March 2007 o 2006 2007 PN 5321001 Revision C Chapter 3 Essential Concepts This section reveals the basic concepts upon which Gen5 was built Learning them will enhance your experience using Gen5 Experiment vs Protocol File Storage Best Practices 44 Chapter 3 Essential Concepts Essential Concepts Understanding the basic concepts behind Gen5 s structure and behavior will help you make the most of it A few topics are covered here on the next few pages more information is provided in Gen5 s Help Experiment vs Protocol on page 45 File Formats Gen5 s files are identified by their filename extension e prt Protocol file e Xpt Experiment file contains the protocol and any data acquired or generated within the experiment File Storage on page 47 Best Practices on page 48 In Gen5 select Help Help Topics to find M ultiple Plate Experiments Security and FDA Electronic Records Compliance Only the Gen5 Secure level of software offers all the capability required to meet the FDA s electronic records requirements 21 CFR Part 11 Experiment vs Protocol Experiment vs Protocol 45 Gen5 uses two common terms to define distinct elements of its toolkit The distinction is subtle and understanding it will improve your Gen5 experience Protocol prt Experiment xpt
8. and enter 10 in thefield then dick in the STD1cell then in the STD2 cell and each subsequent cell in the table until STD8 Click OK to save and close the concentrati ons e Atthegrid set the Number of Replicates to 2 and select Next Conc under Auto Select Click and hold as you roll the mouse over the 5 and 6 columns the cursor changes to a black down facing arrow to fill the entire columns e Set the Well Settings Type to Blank keep the Number of Replicates at 2 and click and drag over wells A 1 and A2 e Set the Well Settings Type to Sample keep the Number of Replicates at 2 and select N ext ID under Auto Select Click and drag the cursor over the remaining wells in columns 1 and 2 and then 3 4 and then 7 12 to assign samples to all the other wells of the plate E Double click Data Reduction Gen5 automatically creates the Blank Subtraction transformations e Click Transformation to set up the calculation Click Select multiple data sets 2 Ze 10 11 12 13 Dual Wavelength Absorbance Endpoint 63 For DS1 selected by default usethe drop down list to select Blank 410 Select D S2 and usethe drop down list to select Blank 630 Click OK to close the M ultiple D ata Set dialog For this example we ll call the New Data Set Name D ual Wavelength Enter the name in the text box Dual wavelength is also known as Delta OD you may want to use this name instead In the Plate Formula field enter
9. e Standards 4 SettheReplicates to 4 5 Assign the well IDs to their corresponding locations in the plate matrix by clicking in the respective wells in the matrix Usethe Auto Select options to speed up your work 4 Definethe Data Reduction E Data Reduction Data Reduction Steps Add Step Description Data Out Comments Jal Transformation Blank 650 Blank Subtraction 5 Curve Analysis Curve tanc Gen5 creates a Blank Subtraction data set when you put blanks on the plate as defined above Click in the white space below the Transformation step e Click Curve Analysis to create a standard curve e Data In Well ID is setto STD Set the Y Axis Blank 650 68 Chapter 4 Assay Examples e Curve Fit Method is set to Linear Regression 5 Definethe Reporting Requirements Save the Protocol Select File Save when you re finished setting up the protocol You ll be able to use this protocol repeatedly to run this assay in an experiment Select File New Experiment and select the protocol when you re ready to run it i e reagents are reconstituted the plate is prepared etc
10. k plate subtraction in your experiment set up an additional Read Step for the blank plate and then create a D ata Reduction T ransformation to subtract the measurements of the blank plate from the samples plate You can insert a Plate in O ut step in the Procedure sequence to first read the blank plate pausethe experiment to pipette samples to the plate and then read the samples plate Step by step procedure 1 Select File New Protocol 2 Double click Procedures to set the reading parameters 1 First create a Read Step for the blank plate enter Blank for the Step Label to easily identify the raw data Description read Blank Plate 405 630 E Plate Out In Load samples Read blank plate Bread 405 630 Eject plate to pipette samples Read samples 2 Add aPlate In Out step to eject the plate to pipette samples standards etc Optionally enter Load Samples in the comment field 3 Finally create a Read Step for the samples plate Y Other steps can be included in the sequence like Set Temperature and Shake if required 3 SetupthePlate Layout to match the distribution of samples and standards or controls 4 Doubleclick Data Reduction to define a transformation Blank Plate Data Reduction 1 Thedialog will contain any automatically generated data reductions Highlight the top most one and dick Transformation to position the blank subtraction as the first calculation 2 Click Select more data sets 3 I
11. mple 57 3 Noticeon the Data In tab the Well ID is setto STD and X Axis Data to Plate Layout Settings The known concentrations entered for Standards are plotted on the X Axis Usethe drop down list for the Y Axis D ata to select Blank nnn wavelength 4 Click the Curve Fit tab depending on your assay you may want to change the curve fit method to 4 Parameters or another option or use Log values on the X or Y axis For now retain the defaults and click the Data Out tab Take note that the D ata Set Name produced from the standard curve is called Conc by default You can change it Click OK to save and close the curve 5 Click Transformation 1 FortheData In usethe drop down list to select Conc 2 Enter a New Data Set Name for the results of this calculation e g Control 3 IntheFormula field enter X CTL1 100 Retain the default setting to Use singleformula for all wells X represents the value of the current well CTL1isthe well ID for the control we assigned in Plate Layout After the plate is read you can return to the Data Reduction dialog to make any needed changes likethe Curve Fit Method Do not change the Data Out or Data Set N ames this would invalidate the data reduction steps that use those data sets 6 Savethe protocol Now you re ready to define your reporting requirements and run the protocol in an experiment 58 Chapter 4 Assay Examples Subtracting Blank Plate Reads To perform a blan
12. mples when you re ready to continue reading the plate 60 Chapter 4 Assay Examples Pathlength Correction Example Hereis an example of the steps required to perform pathlength correction in an ELISA assay In this example we set up an endpoint A bsorbance read subtract Blank wells from all others and transform the data to determine the concentrations of the unknown samples This is the process used to create the Direct Oligo Quantification assay shipped as a sample protocol with Gen5 Q It may be easier to follow these instructions if you have already watched the Gen5 Basic series of online tutorials select H elp gt Tutorials or if you ve completed the learning exercises described in the Getting Started Guide To set up this protocol we ll define the 1 Reading Procedure 2 Plate Layout 3 Data Reductions Reporting Results is the same process for all types of experiments l Defining the reading Procedure This assay example has the simplest read Procedure a single wavelength Absorbance endpoint read 1 Select File New Protocol 2 Select Protocol Procedure 3 Click the Read button and keep the default settings for Detection M ethod Read Type and Read Speed 4 Fill in the checkbox next to Pathlength Correction Optionally dick the 3 dot button to view and modify if desired the test and reference wavelengths used in the process 5 Set the Wavelength Use the drop down list or type the wavelength in the te
13. n assay hereis an example of the steps required to run a quantitative ELISA assay In this example we set up an endpoint A bsorbance read subtract Blank wells from all others plot a standard curve and define a Control to express the samples as a percentage of the control Q It may be easier to follow these instructions if you have already watched the Gen5 Basic series of onlinetutorials select Help gt Tutorials or if you ve completed the learning exercises described in the Getting Started Guide To set up the protocol well define the 1 Reading Procedure 2 Plate Layout 3 Data Reductions Y Reporting Results is the same process for all types of experiments 1 Defining the reading Procedure This assay example has the simplest read Procedure a single wavelength A bsorbance endpoint read 1 Select File New Protocol 2 Select Protocol Procedure 3 Click the Read button and select the wavelength Use the drop down list or typethe wavelength in the text field overwrite the current value 4 Click OK twice to save the Procedure 56 Chapter 4 Assay Examples 2 Defining the Plate Layout This step is critical for the data reduction steps to be defined later Here s the plate layout we need STEZ STDS 0 ST D4 E BLE ETE ETB TET SPLS SPLS SPLS SPLIG SPLIG SPL18 STD4 STD SPL4 SPLA SPL4 rie sre erie SPL17 SPL17 SPL47 D STEZ STEZ SPL2 SPL2 SPL2 SFL10 SPLIO SPLIO SPL1S SPL
14. n5 s Help Review the description of the sample protocol to make sure it is compatible with your reader 54 Chapter 4 Assay Examples How do I set up my assay Here are step by step instructions for creating Gen5 Protocols to run common assays More Assay Examples can be found in Gen5 s Help We hope that by following the instructions making some changes to names and other details you can adapt them for use in your lab Also seetheKinetic Analysis chapter Absorbance e Quantitative ELISA Example on page 55 e Subtracting Blank Plate Reads on page 58 e Pathlength Correction Example on page 60 e Dual Wavelength Absorbance Endpoint on page 62 e Basic Spectrum Analysis on page 64 e Protein Quantification Assay on page 66 e Max Binding Determination on page 83 e Toxicity Cytotoxicity Assay on page 86 e Endotoxin Test on page 89 e s Galactosidase Kinetic Assay on page 92 Fluorescence e Basic Fluorescence Assay on page 69 e Kinetic Fluorescence Assay on page 71 e Fluorescence Assay with Injection on page 74 e Fluorescence Area Scan Example on page 77 e Fluorescence Polarization on page 79 Luminescence e Basic Luminescence Glow Assay on page 81 e Luminescence Flash Assay with Injection on page 82 Dispensing Reagent e Dispensing Reagent in a Kinetic Analysis on page 94 e Dispensing Reagent in an Endpoint Analysis on page 95 Quantitative ELISA Example 55 Quantitative ELI SA Example To help you set up your ow
15. ne tune the protocol within an experiment but remember to select File gt Save Protocol As to update the original protocol with your improvements Just like word processing documents when you run similar types of experiments you can use File gt Save As to give you a head start creating a new protocol based an existing protocol that contains the same plate layout reading parameters or other elements that will be repeated in your new protocol Define and customize Data Views before selecting what to include in reports or export files All the on screen data i e data views can be reported or exported If you use on screen views and paper reports equally it is most efficient to first fine tunethe Data Views and then include them in reports exports Always assign Blanks to the plate Blanks can be deionized DI water buffer reagent without analyte substrate and so on When running fluorescence cellular assays a DI water blank illustrates the background contributed by the instrument and labware as separate from the cells and media Identify the location of the Blanks in the Plate Layout and Gen5 will automatically create the blank subtraction data reductions Backup your database regularly BioTek recommends once per week for most organizations If you re using Gen5 s Database for protocol and experiment file storage usethe built in Periodic Optimization feature Take action if you get a warning message about the remaining size of your
16. nt select File New Experiment 5 Viewing the Results After you read the plate you can take advantage of Gen5 s Well Zoom to examine the results This feature is described in the Kinetic Analysis chapter 66 Chapter 4 Assay Examples Protein Quantification Endpoint Absorbance Here are instructions for the Gen5 portion of running this type of assay the easy part Correctly mixing and dispensing the standards and pipetting reagents to the plate isthetricky part Follow the assay instructions dosely and modify these steps as needed Click the links for instructions at each step 1 Select File gt New Protocol 2 Definethe Procedure E Procedure Synergy Com1 Add Step Plate Type 06 WELL PLATE Read Description Dispense W Read 650 Shake 1 El Select Protocol Procedure 2 Click Read to add one read step 3 Keepthe default settings e Detection Method Absorbance e Read Type Endpoint 4 SettheWavelength using the drop down or enter 650 in the nm field 5 Click OK twiceto close and save the Read step and the Procedure Protein Quantification Endpoint Absorbance 67 3 Definethe Plate Layout 1 2 3 STb1 STb1 STb1 Set up Gen5 s plate layout to match your placement of samples and standards on the plate for example l ui Select Protocol Plate Layout 2 IntheWell Settings box select the Type of specimen first Standards then Blanks then Samples 3 z Define the Concentration of th
17. ntheMultiple Data Sets screen use the drop down lists to select the Blank plate read data for DS1 and the samples plate data for DS2 In multiple wavelength protocols there may be several data sets to choose from If you ve used the Step Labels for each Read Step it s easy to match up the blank plate with the samples plate Subtracting Blank Plate Reads 59 Data Sets Plate Data In O pst zCurrent Plate gt gt blank 410390 E 2 DSZ Current Plate E EEE blank 41390 blank A3630 CD54 test 41 590 test Az 410 4 EnteraNew Data Set Name for the resulting data set e g Blanked 390 Enter DS2 DS1 in the Plate Formula field and click OK 6 Repeatsteps 2 4 to create as many blank plate subtraction Transformations as needed e g one per wavelength 7 Now you re ready to create other Data Reduction steps using the blanked data sets For example select Curve Analysis to generate a standard curve based on the blank subtracted test plate 5 Customize the Data Views and fine tune the Report Builder as needed before saving and closing this protocol File gt Save Now you re ready to run the protocol in an experiment File gt New Experiment Running the experiment After reading the blank plate Gen5 ejects the carrier so you can load the samples A Load Samples If you entered a comment e g Load Samples in the Plate In Out step it is displayed on screen H ere s how to proceed e Click OK after loading the sa
18. ons for programming commonly known assays using Gen5 Gen5 also ships with Sample Protocols for most of these assays Both the Sample Protocols and the assay descriptions are learning tools It is your responsibility to customize and validate them to meet your needs Sample Protocols and Experiments sese 52 How do set up my assay sssssssssesee emen 54 52 Chapter 4 Assay Examples Sample Protocols and Experiments Numerous sample protocols are shipped with Gen5 You can usethe protocols to learn more about Gen5 and as a timesaver customizing them to meet your needs and then running them in an experiment to obtain results Recommendation Before making any modifications to the sample protocols we recommend selecting File gt Save As after opening them and assigning a unique name to the protocol This will preserve the original sample protocol for future use A matching experiment file is also shipped with Gen5 for use as a learning tool M any of the experiment files contain actual data so you can see how Gen5 presents the results on screen and in reports a Find the sample protocols and experiments shipped with Gen5 in the default file storage locations A folder for each detection method is available Absorbance Fluorescence Luminescence and for Synergy 2 users there is a Synergy 2 folder within each detection method folder e Gen5 Secure and database users Select File Open Protocol in the DB
19. orials or if you ve completed the learning exercises described in the Getting Started Guide l Defining the reading Procedure This assay example uses a kinetic read for analysis 1 Select File New Protocol Open the Procedure double click Procedure in the menu tree Click Read and change the Read Type to Spectrum Set the Wavelength range for this exercise set Start to 200 and Stop to 550 Set the Step to 3 and close the Read step er XL e me XV Click OK to save and close it 2 Defining the Plate Layout Definethe platelayout in the usual way to reflect the arrangement of unknown samples standards and blanks on the microplate 3 Defining the Data Reduction Steps Now that you ve defined the reading parameters and plate layout you can definethe data reduction steps 1 Select Protocol Data Reduction Gen5 automatically sets up the a Well Analysis for Min Max OD If Blanks have been assigned to the plate it will be preceded by and based on a blank subtraction Transformation step Click Well Analysis to add another step Enter a unique name for this step in the Label field Select one of the offered Calculation Types Integral or Formula Click OK to save and close the step Ss eS E MES Click OK to save and close Data Reduction Basic Spectrum Analysis 65 4 Save the Protocol 1 Define the Reporting Requirements using the Report Builder or export options 2 Save the protocol Now you can run itin an experime
20. the protocol development stage but it uses the default protocol and generally requires reading parameters to be defined Multiple plates can be processed in an experiment each one considered a unique assay with independently reported or exported results The exception is multi plate protocols described later Xpt is the experiment s filename extension An experiment is saved as the full collection of procedures formulas reporting definitions and other details i e the protocol and the plate data from readings and calculation results Data acquired and transformed in an experiment is protected by an audit trail in both Gen5 Secure and other Gen5 software editions The Reader Control edition does not support this feature 46 Chapter 3 Essential Concepts Protocol prt Experiment xpt Changes made to a standalone protocol Within an experiment you can select are not reflected in any previously Save Protocol As to capture the current created experiments based on that details of the protocol and save them as protocol A new experiment must be either a new protocol or as an overwrite created to apply the revised protocol of the original protocol Y All newly created protocols and unless another protocol is selected experiments are based upon the Default Protocol Gen5 also supports more complex multi plate protocols that are not covered in this introductory material Check out Designing a M
21. ulti Plate Protocol in a subsequent chapter About File Storage 47 About File Storage File Types Gen5 creates two file types Protocol prt and Experiment xpt e TheGen5 executable file exe and numerous other types of supporting files like an Excel template are also installed on the computer e n addition Clarity Microplate Luminometer users will work with Clarity protocol files which use a BPF extension Gen5 references the Clarity files as they contain the reading parameters required to control the luminometer Databases Gen installs two databases on your system called LocalDB and SharedDB While the databases are always used for critical internally used files Gen5 offers you the choice of using the Windows File System or the Gen5 SharedDB database for storing Gen5 s Protocol prt and Experiment xpt files This option combined with the ability to create multiple databases allows you to structure file storage according to your organization s requirements e Files may be stored on the computer s hard drive on a network or on a CD or other portable medium Windows Explorer or a similar application can be used to view the file names and locations and to move copy rename and delete files e Alternatively protocol and experiment files may be stored in a secure shared access database This database initially named SharedDB mdb can be stored on a user s computer or on a shared access network comp
22. uter LAN Gen5 provides a special file maintenance utility for viewing thefile names and their locations and for moving copying renaming deleting importing and exporting files e Select the preferred method of storing protocol and experiment files at System Preferences File Storage File Location During a conventional installation e the program files are stored in this default location CA Program Files BioTek Gen5 software edition e the databases are stored in these default locations Windows XP and 2000 systems CA Documents and Settings All Usera Application Data BioTek Instrumental Gen5 software edition version SharedDB or LocalDB Windows Vista Windows XP and 2000 operating systems CA Program Data BioTek X Genb software edition version SharedDB or LocalDB e Gen5 installs Protocol and Experiment folders in the respective File Storage locations e g CA Program Files BioTek Gen5 software edition Protocol 48 Chapter 3 Essential Concepts Best Practices Like most software tools Gen5 is flexible it offers several ways to accomplish a task H ere are some recommendations for saving time and using it most efficiently Efficiencies For an assay or experiment that you will run numerous times develop a Protocol to define the Procedure Data Reduction Data Views and Reports required Then you can run an Experiment File gt N ew Experiment based on the Protocol whenever necessary Y ou can fi
23. xt field overwrite the current value For this example enter 260 6 Click OK twiceto savethe Read step and the Procedure 2 Defining the Plate Layout For this assay example the platelayout is very simple comprising two blank wells and 94 unknown samples gt Select Protocol Plate Layout Pathlength Correction Example 61 In the Well Settings box select the T ype of specimen first Blanks then Samples Assign the blanks to cells A1 and B1 Change the T ype to Sample make sure N ext ID is enabled click and drag over the remaining wells to assign the unknown samples 3 Defining the Data Reduction Steps After defining the reading parameters and plate layout we can define the data reduction steps Gen5 creates the blank subtraction and the pathlength correction for you automatically l 6 Select Protocol Data Reduction Notice thetwo Transformations Blank 260 and Corrected Blank 260 Gen5 first subtracts the blanks and then applies the Pathlength Correction Calculation Click Transformation to add anther Data Reduction step For the Data In usethe drop down list to select Corrected Blank 260 data set Enter a New Data Set Name for the results of this calculation e g Concentration In thePlate Formula field enter X 32 5 Retain the default setting to Use single formula for all wells X represents the value of the current well The extinction coefficient for ssDNA oligonucleotides 1 mg ml at
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