iq 5 Calibration: Contents.

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1 iq 5 Calibration: Contents. When do you calibrate Preparation. What you will need Assembly. What you will prepare Procedure. What you will do Summary Detailed Procedure Mask Background Well Factors Pure Dyes Calibration Files Format and Naming Conventions Locations Viewing Calibration Files Evaluating Calibration Files Masks Background Well Factors Pure Dyes RME Format Identifying Calibrated Fluorophores Fluorophore Reference Tables Using the iq5 RME (Pure Dye) Decoder Evaluating RMEs SYBR Green is pre-calibrated Multiple FAM probes (or other fluors) Improper Calibrations on the iq5 Components and Part Numbers 1

2 When do you calibrate: You should Calibrate the iq5: 1. When the unit is installed. 2. If any component that affects the light path is replaced. These include: a. Lamp b. Filters c. Optical Lid d. Optical Unit e. CCD camera (service visit/depot repair only) 3. You replace the PC running the instrument and you have lost the calibration files. It is easy to backup the calibration files (see page 10, Calibration File Locations) 4. Whenever prompted by the software. Background factor and Persistent Well Factor calibrations contain a 30 day timer and the.software will display a warning when starting a run with outdated background and persistent well factor calibrations. What you will need: Obtain the following components: 1. icycler iq Calibrator Dye Solution Set 2. icycler iq External Well Factor Solution (found in the iq Calibrator Dye Solution Set) 3. 3 x 96-well PCR plate or preferred reaction vessel 4. Optical quality sealing tape or preferred sealing method Make the following decisions: 1. What volume you want to calibrate. Other than Background calibration all calibrations should be done using the volume used in experiments. 2. What reaction vessels you wish to calibrate. All calibrations require you identify and use a specific vessel and seal type. 2

3 What you will prepare: Prepare three plates (or tube formats) and label them as indicated below: 1. Plate A : 96 wells filled with well factor solution. a. Dilute the 10x External Well Factor Solution to 1x with water. b. Pipette equal volumes of the 1x external well factor solution into ALL 96 wells using the volume that will be used in your experiment. c. Seal the plate using the sealing method you will use in your experiments. 2. Plate B 96 empty vessels of choice (air NOT WATER). 3. Plate C Pure dyes. a. Pipette equal volumes of the appropriate 1x calibrator solution into 8 wells (usually by column) of the 96-well plate, using the volumes that you will use in your experiment. b. Repeat for all fluorophores needed. c. Seal the plate using the sealing method you will use in your experiment. 3

4 Summary of what you will do: The following is a list of the four calibrations followed by plate (or tube format) used to calibrate. 1. Mask 96 wells filled with well factor solution (Plate A ) 2. Background - 96 empty vessels (Plate B ) 3. Well Factors - 96 wells filled with well factor solution (Plate A ) 4. Pure Dyes One column or row of wells filled with each pure dye to be calibrated. (Plate C ) Each calibration step has a dedicated tab in the Calibration module. Calibrations should be performed in the sequence in which they are appear in the software (1) Mask, (2) Background (3) Well Factors (4) Pure Dye. 4

5 Detailed instructions: Turn on the instrument at least 10 minutes before performing a calibration procedure. Mask Alignment: Use numbers in image to find controls for the associated steps: 1. Load 96 well factor plate into the iq5 instrument (Plate A) 2. Navigate to Calibration Module and the Mask tab. 3. Click Home. 4. Click the Filter Position 3 option button. 5. Click Take an Exposure. 6. Click Show Mask. 7. If any pink pixels are present in the mask, reduce the exposure time and retake an exposure. Keep retaking the exposure until no pink pixels are present. 8. Click Optimize Mask. 9. Click Save Mask. Notes on Mask Calibration: 1. IF MASK ALIGNMENT FAILS press Restore Factory Mask and adjust the mask to more closely match the well configuration in the image. The following controls are near the number 8 in the above image. a. Use the + and buttons to adjust well spacing 5

6 b. Use the Up, Down, Left and Right buttons to orient mask array over the wells in the image. 2. The optimize mask function may benefit from repeat iterations because the mask will only move a certain number of pixels each time it is oriented. However, it is unlikely more than two iterations will be necessary. 3. Valid masks must be saved before performing other calibrations. 4. The Optimize Mask function is MUCH better than the human eye. If you ve pressed this button several times the mask is optimal. Save it and move on. Background Calibration: 1. Load the empty vessels (Plate B) in the iq5. 2. Select a well seal. 3. Select a vessel type. 4. Click Collect Background Factors. 5. When iq5 completes the Background Calibration run, a dialog box appears with the message, "Background Calibration Run Complete." 6. Click OK to exit. 6

7 Persistent Well Factor Generation: 1. Load the well factor vessels (Plate A) in the iq5. 2. Select a sample volume. 3. Select a well seal. 4. Select a vessel type. 5. Click Collect Persistent Well Factors. 6. When iq5 completes the Persistent Well Factor Calibration run, a dialog box appears with the message, "Persistent Well Factor Calibration Run Complete". 7. Click OK to exit. 7

8 Pure Dye Calibration: 1. Load the Pure Dye vessels (Plate C) in the iq5. 2. Select (a) or Create (b) a Pure Dye Plate Setup. a. Select existing calibration plate from the User 1 folder or use the default plate setup. At install you will use the PureDyeCalibrationKit plate setup. Make sure your Sample Volume, Seal Type, and Vessel Type match your calibration run. (#3 in figure for 3b) b. Choose Create New 1.) Select/Add Fluorophores to add the desired fluors to your calibration plate. 2.) Choose or add fluorophores which will be loaded into the plate locations in which you have loaded the corresponding calibration solution. 3.) Choose Sample Volume, Seal Type, and Vessel Type. 4.) Save and Exit (to the right of 3 in the following figure) Custom Fluorophores are added here. You must know which filter set is optimal for the new fluorophore. 8

9 3. Click Run Pure Dye Calibration. 4. When iq5 completes the Pure Dye Calibration run, a dialog box appears with the message, "Pure Dye Calibration Run Complete". 5. Click OK to exit. You can calibrate the iq5 for as many fluorophores as you can fit on one plate in one calibration run. There are no restrictions associated with fluorophores read through the same filter. 9

10 Calibration Files: Format and Naming Conventions: All calibration files are saved in an xml format. Most systems will launch internet explorer to view these files. There files are highly formatted text. All Calibration files are automatically named using the following conventions. Bold font indicates a variable component of the name that will match the settings chosen for the particular calibration. Masks: There is only one mask file and it is named mask-data. Background: Background_Vessel_Seal_iQ5 Serial #_Base Unit Serial # There will be one active background factor file for each vessel/seal combination that is calibrated. During a run the software will automatically use the correct file for the parameters specified in a plate setup. Well Factors (persistent): Persistent_Vessel_Seal_Volume_iQ5 Serial #_Base Unit Serial # There will be one active well factor file for each vessel/seal/volume combination that is calibrated. During a run the software will automatically use the correct file for the parameters specified in a plate setup. Pure Dye: RME_Vessel_Seal_Volume_iQ5 Serial #_Base Unit Serial # There will be one active well factor file for each vessel/seal/volume combination that is calibrated. During a run the software will automatically use the correct file for the parameters specified in a plate setup. The following is an example of a typical RME name: RME_Plates_Film_25ul_575BR 0123_br RME indicates that it is a Pure Dye file Plates indicates that this file was generated using plates Film indicates that this file was generated using film 575BR 0123 is the serial number of the Optical unit br indicates a base unit serial number of 582BR

11 Calibration File Locations (Folders you should back up): Each active calibration file is located in its own folder in the iq5 directory. After calibration these folders can be backed up on reliable media. They can then be retrieved if you replace the computer or if there is a fatal computer malfunction. Masks C:\Program Files\Bio-Rad\iQ5\Masks Background C:\Program Files\Bio-Rad\iQ5\BackgroundFactors Well Factors C:\Program Files\Bio-Rad\iQ5\WellFactors Pure Dye C:\Program Files\Bio-Rad\iQ5\RMEData Background Factors Persistent Well Factors and RME (Pure Dye) calibration files from previous calibrations are archived in subdirectories called Backup located in the associated calibration folder. Ex. C:\Program Files\Bio-Rad\iQ5\RMEData/Backup RME Archived calibration files can be viewed in the same manner as outlined below for the active calibration files. Viewing Calibration Files. The iq5 software has an embedded converter that will convert all calibration files with the exception of Masks to into a readable format. To view any of these calibration files (1) go to the View Menu and choose Calibration Data. (2) Navigate to the appropriate folder for the file you are interested in viewing. Note that the active calibration files will be located in the directories listed above. 11

12 Evaluating Calibration files Evaluating Masks: The best way to evaluate a mask is to take an image of a well factor plate and evaluate the current mask against that image. Trust the Optimize Mask algorithm. If you have pressed the Optimize Mask button several times the mask in the image window is the best possible one for the instrument. Do not attempt to align a mask manually. It is not useful to open and view the actual xml mask file. Like all calibration files the mask tracks the serial number of the Optical Module and thermal cycler. iq5 will not use a mask in which the embedded serial numbers do not match that of the instrument. Evaluating Background Factors: Background files can be opened with the embedded xml viewer as outlined above. The data is presented in two formats. The first is a list of values for each well with a column for each filter set. Below this the data is presented in a plate layout for each filter set. This makes it easy to evaluate the location of high or low spots. Background values are usually single or double digit numbers and should be fairly consistent across the plate. Evaluating Well Factor: Well Factor files can be opened with the embedded xml viewer as outlined above. Just like background factors well factor data is presented in two formats. The first is a list of values for each well with a column for each filter set. Below this the data is presented in a plate layout for each filter set. This makes it easy to evaluate the location of high or low spots. The software considers well factor values from 0-4 valid. 12

13 Evaluating Pure Dye Files (RME, Response Matrix Element): RME Format (when opened in iq5 Calibration File viewer): RME files can be opened with the embedded xml viewer as outlined above. The data is presented in two formats. The first is a complete list of values for each fluorophore that has been calibrated with a column for each filter set. Below the comprehensive list the data is presented in an individual table for each fluorophore. Identifying Calibrated Fluorophores. If a fluorophore is not present in the Summary table then the iq5 in question has not been calibrated for this fluorophore. Fluor ID: Each Fluorophore is assigned an ID number by the software. Except for custom fluorophores this ID number is consistent from instrument to instrument. For instance FAM will always be Fluor ID number 9. The following is a list of standard fluorophores, their ID # s and the filter set through which signal is read: 13

14 Fluor IDs sorted by ID #. Fluorophore Fluor ID (Number in RME File) Excitation Emmission Read Through Filter Set Cy /25X 595/24M 4 FAM 9 490/20X 530/30M 2 HEX /30X 575/20M 3 TET /30X 575/20M 3 VIC /30X 575/20M 3 TAMRA /25X 595/24M 4 JOE /30X 575/20M 3 TexasRed /30X 620/30M 5 ROX /30X 620/30M 5 Cy /30X 680/30M 6 LC /30X 680/30M 6 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 Fluor IDs sorted alphabetically by Fluorophore Name Fluorophore Fluor ID (Number in RME File) Excitation Emmission Read Through Filter Set Cy /25X 595/24M 4 Cy /30X 680/30M 6 FAM 9 490/20X 530/30M 2 HEX /30X 575/20M 3 JOE /30X 575/20M 3 LC /30X 680/30M 6 ROX /30X 620/30M 5 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 SYBR /20X 530/30M 2 TAMRA /25X 595/24M 4 TET /30X 575/20M 3 TexasRed /30X 620/30M 5 VIC /30X 575/20M 3 14

15 iq5 RME (Pure Dye) Decoder: A simple excel worksheet is available for troubleshooting RME files. This worksheet will allow you to view the list presented in the RME file with the fluorophore names so that you do not have to continually cross reference with the above charts. It will also identify the assigned read through filter set for each fluorophore and aid you in evaluating the calibration data. To use this excel document 1. In iq5 go to "View" Menu 2. Choose "Calibration" data. 3. Navigate to "RMEData" folder for resident RME or to the RME in question if you are working remotely. 4. Open the RME you wish to inspect. 5. Highlight and Copy every cell of the summary table. (first table) 6. Click in well A1 of the sheet named Paste Data Here (should be the first you see) and paste. 7. Navigate to the Decoder Sheet. Column G will now contain the name of the fluorophore based on the Lookup sheet. (Which is current as of iq5 v 1.0) 15

16 Note that the Decoder sheet should now look something like the following table. Fluor ID Filter Position Fluor Name Read through Filter Set SYBR LC Cy ROX TexasRed JOE TAMRA VIC TET HEX FAM SYBR SYBR SYBR SYBR SYBR Cy #N/A #N/A #N/A Evaluating RMEs: Generally the calibration value for each fluorophore should be highest through its assigned filter set. The iq5 RME Decoder can aid you in evaluating this. Next to the fluorophore name the filter set the fluorophore signal is read through is indicated. The spreadsheet automatically highlights the highest value for each fluorophore green. There are a few other sheets in the workbook. 1. Directions. In case you cannot see the embedded comments. 2. Lookup sheet. This is where the workbook is getting the information to decode the Fluor IDs. Note the key that indicates where you can edit for custom fluorophores. 3. Two reference sheets which list Fluor IDs and names. One is for current fluorophores and one is a list of legacy fluorophores which reside in the iq5 software to support data files produced on previous software packages. 16

17 SYBR Green Calibration: It is unnecessary to calibrate for SYBR Green I. The software uses a hard coded value for SYBR green, which includes the multiple versions of SYBR that are present in order to run multiple SYBR assays in one experiment. Don t calibrate for SYBR or be concerned about SYBR Green calibration files in the RME. The software is using values embedded in the code for SYBR green data collection. Multiple FAM (or other fluorophores): Customers may want to run multiple FAM probes on each plate and use the plate setup to track their locations. This can be accommodated by calibrating for multiple instances of the FAM fluorophore and naming each incidence differently (FAM1, FAM2, etc). If a customer does not want to perform these calibrations he may use the multiple SYBRs in place of multiple FAMs. 17

18 Improper Calibrations on the iq5 Why Ensure Proper Calibration of the iq5? Each of the iq5 s four calibrations is important. Skipping or cutting corners around calibration may result in sub-optimal results. Mask: A poorly aligned mask may result in the collection of invalid or inconsistent data and would typically result in poor replicate reproducibility. The iq5 camera sends data in the form of plate images to the computer. The iq5 software utilizes the mask to determine the exact bounds and coordinates of each well within the plate image. Background Factors: Improper background calibration can affect both replicate reproducibility and spectral deconvolution in a multiplex experiment. An empty PCR plate or tube does not yield a fluorescence of zero, but does in fact generate a quantifiable amount of signal. Background Factors account for this signal. Without proper background factor calibration, signal generated by the vessel and sealing mechanism may be inaccurately attributed to the fluorescent chemistry, resulting in suboptimal optical data. Stability tests on background factors are in progress. It is possible that in the future the recommended time between calibrations will be much longer. Well Factors: Improper persistent well factors may result in sub-optimal uniformity and larger replicate spread. Although the iq5 does take relatively clean and uniform images, there are slight well-to-well differences characteristic of each optical system. The iq5 exhibits excellent well-to-well uniformity when well factors have been collected properly. Stability tests on well factors are in progress. It is possible that in the future the recommended time between calibrations will be much longer. 18

19 Pure Dye Calibration: Failure to run a valid pure dye calibration may result in dramatic base line drifts and other strange results when running multiplex experiments. Most fluorophores contribute a significant amount of signal to more than just one filter pair, i.e. there is signal overlap or bleed-through into different filter pairs. The pure dye calibration takes this into account by measuring the signal of each fluorophore, alone, through each filter pair used. In multiplex, the iq5 software uses the pure dye calibration file to calculate the total signal generated by each fluorophore. Components/parts used in this procedure: icycler iq Calibrator Dye Solution Set icycler iq External Well Factor Solution well 200ml Thin Wall PCR Plates, 25 per box Optical Quality Sealing Tape, optimized for use with , 100 sheets well 200ml PCR Plate Caps, for , 300 per box Other components of interest: icycler iq FAM Calibrator Solution, 5 per package icycler iq HEX Calibrator Solution, 5 per package icycler iq Texas Red Calibrator Solution, 5 per package icycler iq Cy5 Calibrator Solution, 5 per package Replacement Halogen Lamp iq5 Software Installation Disk 19

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