Spectral Calibration page
Contents
1. If you then change or eliminate one of these reporters in your assay it is strongly recommended that you recalibrate your instrument for this new arrangement 4 For each custom dye to be calibrated prepare solutions by diluting into PCR buffer to the appropriate concentration above table and a final volume of 125 uL This should leave you with 25 uL excess Also prepare 125 uL of PCR buffer alone Ensure that each of these solutions is uniformly mixed 5 Dispense 25 uL of each dye solution into each of four reaction tubes Do the same for the PCR buffer Label the tubes accordingly and spin them down in the minifuge so that the solution collects into the Spectral Calibration FINAL 092305 doc d l Page 9 of 12 S BIOSEARCH TECHNOLOGIES Chemistry for Genomics and Proteomics diamond reservoir If any of these tubes have bubbles remaining in the reservoir spin them down again Set these tubes aside for future use 6 Turn on the Smart Cycler II instrument and open the Smart Cycler application From the Tools drop down menu select Optical Calibration 7 From the Dye Set Location drop down menu select one of the two user accessible entries to calibrate either Dye Set 5 or Dye Set 6 Type a name for this dye set to remind you which dyes were calibrated and the volumes involved For example a 25 uL volume calibration for the dyes FAM CAL Fluor Orange 560 CAL Fluor Red 610 and Quasar 670 could be titled FORQ25 8 Select a Hold Temp2. CA iCycler and Opticon 2 are registered trademarks of Bio Rad Laboratories Hercules CA SmartCycler and SmartCycler II are registered trademarks of Cepheid Inc Sunnyvale CA Spectral Calibration FINAL 092305 doc
3. and a Read Temp that is identical to the temperature you will be acquiring fluorescence data during your actual real time PCR amplifications For a typical TaqMan 2 step reaction this is 60 C Select the 25 uL tube size The Hold Time and the Read Time can be left at 10 seconds each 9 Inthe Dyes pane type acronyms for the names of the dyes you will be calibrating across the four channels and the concentration associated with each FAM should be calibrated on channel 1 CAL Fluor Gold 540 CAL Fluor Orange 560 CAL Fluor Red 590 and Quasar 570 should all be calibrated on channel 2 CAL Fluor Red 610 should be calibrated on channel 3 CAL Fluor Red 635 and Quasar 670 should be calibrated on channel 4 If any channels will be left blank such as for a duplex or triplex assay type unused or empty into the Name field of the appropriate channel _ In the Excitation Emission Mapping pane make sure that the wavelength range associated with each channel is correctly positioned for the associated dye However if any channel will be left un calibrated the excitation and emission mapping for that channel should be left at its default values Otherwise channel 1 should have an excitation of 450 495 nm and an emission of 510 527 nm channel 2 should have an excitation of 500 550 nm and an emission of 565 590 nm channel 3 should have an excitation of 565 590 nm and an emission of 606 650 nm and channel 4 should have an excitation o
4. Page 1 of 12 lt BIOSEARCH SS TECHNOLOGIES Chemistry for Genomics and Proteomics CAL FLUOR AND QUASAR DYES Thermal Cycler Spectral Calibration Instructions WHY IS SPECTRAL CALIBRATION NECESSARY In order to successfully use dual labeled probes containing CAL Fluor and or Quasar dyes in real time quantitative PCR qPCR multiplexed assays certain real time PCR instruments must first be calibrated to recognize the pure dye spectra Although unnecessary for simple singleplex experiments spectral calibration is critical for multiplexed assays so that overlapping fluorescent signals can be resolved from one another This document provides detailed step by step instructions for calibrating these instruments REAL TIME INSTRUMENTS CURRENTLY SUPPORTED REAL TIME INSTRUMENTS NOT SUPPORTED ABI Prism 7700 ABI Prism 7000 Bio Rad iCycler ABI Prism 7300 Cepheid SmartCycler II ABI Prism 7500 Bio Rad MJ Opticon 2 ABI Prism 7900 Cepheid SmartCycler EMISSION SPECTRA OF CAL FLUOR AND QUASAR DYES o wo E a o J EE FAM CAL Fluor Gold 540 EE CAL Fluor Orange 560 GS Quasar 570 EE CAL Fluor Red 590 CAL Fluor Red 610 E CAL Fluor Red 635 E Quasari 670 o mn normalized fluorescence o a BJ lid b LA o pa 0 Wavelength nm Spectral Calibration FINAL 092305 doc EO Page 2 of 12 TECHNOLOGIES Chemistry for Genomics and Proieomi CALIBRATION DYE TUBE CONTENTS Ea
5. ate containing the calibration dye s Spectral Calibration FINAL 092305 doc BIOSEARCH Page 7 of 12 SS TECHNOLOGIES T z 1 A t PEMTGIFY for Genomics and Proteomics 9 xr xr Nr Switch to the Select and load fluorophores pane and deselect any of the fluorophore entries that are currently checked The fluorophore entries are labeled with standard dye names followed by the excitation filter through which they are detected Make a note of those standard dye entries that are coupled with excitation filters appropriate for the Biosearch dye s you will be calibrating Ideally your choice should be an entry that is infrequently used and whose calibration information can be overwritten to accommodate the Biosearch dye CAL Fluor Gold 540 CAL Fluor Orange 560 and Quasar 570 are each optimally excited using the 530 30X filter CAL Fluor Red 590 is optimally excited using the 545 30X filter CAL Fluor Red 610 is optimally excited using the 575 30X filter CAL Fluor Red 635 and Quasar 670 are each optimally excited using the 635 30X filter IMPORTANT NOTE the iCycler does not permit you to create new dye entries beyond their list of standard dyes Therefore to use a custom dye on this instrument requires calibrating a standard entry using the custom dye calibration solution It will be a good idea to leave a note near the instrument outlining the dyes that have been calibrated for the various entries This calibration will o
6. ch calibration dye is supplied in an individual tube containing 5 nmol of lyophilized calibration dye covalently linked to a 10 base oligonucleotide comprised of poly T residues T10 Biosearch Provides Calibration Dyes in the specifications shown below Calibration Dye Product Number Amount Provided USPrice pram RAH mol 95 00 CAL Fluor Red 635 RD 5084 95 00 Quasar 570 RD 5063 95 00 Quasar 670 RD 5065 95 00 CALIBRATION CONCENTRATIONS Commercially available real time thermal cycler models differ substantially in the sensitivity of their optics Furthermore several instruments of the same model can also differ in fluorescence detection sensitivity Calibration concentrations required for each of the CAL Fluor and Quasar dyes will therefore vary according to instrument Recommended concentrations determined empirically at Biosearch and with selected scientific collaborators working in the field are provided within the instrument specific instructions included below We suggest that you fine tune these values using the procedure specified by your instrument manufacturer If your model is not included in this document please consult the instruction manual that came with your instrument for guidelines on custom dye calibration PREPARATION FOR USE After determining the final working concentration within your instrument specific instructions create a stock solution at a higher concentration by re suspending this calibrati
7. concentrations based on the characteristics of your specific instrument aiming for a consensus concentration that achieves a gain setting of 2 across most reaction sites NO amp O JOJO N 1 These calibration instructions are specific to 25 uL reaction volumes If you use different reaction sizes the recommended concentrations listed in the above table may no longer be appropriate for your application and the instructions will need to be adjusted accordingly 2 The SmartCycler Operator Manual recommends preparing sufficient tubes to simultaneously calibrate all reaction sites on a processing block 16 tubes for each custom dye and an additional 16 tubes containing only buffer the instruction to prepare 16 tubes that contain a mixture of all dyes is specific to the SmartCycler but unnecessary for the SmartCycler II With the possibility that our recommended concentrations are unsuitable for your specific processing block we recommend only preparing enough tubes to calibrate four reaction sites at a time using these same tubes to sequentially calibrate the remaining sites Using this strategy to calibrate for a quadraplexed assay would only require 16 tubes in total and if one dye proves to be inappropriate only four tubes would need to be prepared at a new concentration 3 All dyes that you plan on using simultaneously as reporters in a multiplexed assay will need to be calibrated in the same run to produce a single dye set
8. er doing so click Go 7 Once the measurements have finished again inspect for anomalous results The signal detected by the dye in at least one of the channels should be over 10X the signal produced by the blank plate in that channel Importantly the ratio of intensities of channel 1 to channel 2 should be consistent across the plate After inspection click Next Spectral Calibration FINAL 092305 doc i SOSEARCH Page 12 of 12 TECHNOLOGIES T z 1 A t PEMTGITY for Genomics and Proteomics 8 Click Finish and the calibration procedure is complete To repeat the procedure for any additional custom dyes that you wish to calibrate on this instrument click Yes at the final prompt LEGAL DISCLAIMERS TRADEMARK amp COPYRIGHT INFORMATION Copyright Information 2005 Biosearch Technologies Inc CAL Fluor amp Quasar Trademarks and Applications Information CAL Fluor Gold 540 CAL Fluor Orange 560 CAL Fluor Red 590 CAL Fluor Red 610 CAL Fluor Red 635 Quasar 570 and Quasar 670 are trademarks of and licensed by Biosearch Technologies Inc Novato CA for research and development use only Their use in human or veterinary diagnostics applications is strictly prohibited Commercial licensing opportunties are available Please contact the Director of Marketing Sales and Licensing at licensing biosearchtech com for additional information PCR Technology PCR is a proprietary technology covered by several US patents including US Patent N
9. f 680 650 nm and an emission of 670 750 nm 11 Click the Select Sites button at the bottom of the window and select the first four reaction sites that you will be calibrating typically A1 A4 Click OK 12 Click the Start Calibration button at the bottom of the window and click Proceed at the prompt that follows 13 Anew popup window will appear prompting you to place the four buffer only tubes into the appropriate reaction sites After doing so click OK Four LED s associated with the reaction sites you are calibrating should light up 14 After fluorescence readings of the buffer tubes have been obtained the instrument will prompt you to replace these tubes with those containing your first calibration dye After doing so click OK Follow the instrument prompts for the remaining dyes you will be calibrating Each time you place and remove tubes from the instrument use care in handling so that the sides of the tubes are not scratched and bubbles are not introduced into the diamond reservoir These same tubes will be required later in the procedure If instrument calibration was successful you will be prompted with the choice to either begin or skip the verification process This process confirms that the signal intensity of each fluor is as expected and that bleed through into adjacent channels can be successfully subtracted out It is recommended that you conduct verification after calibration Calibration can fail if either
10. front of your dye acronym ensures that this custom entry will be appended to the end of the list and not prevent the software from recognizing the previously mentioned dyes Make sure that the Quencher box is unchecked Create entries for any additional Biosearch dyes you will calibrate on the same reaction plate and then exit Sample Type Setup by clicking OK In the plate layout select the wells that correspond to the wells in your reaction plate containing your dye calibration solution Once these wells are selected label them as the appropriate Biosearch Pure Dye that is now an entry in the Sample Type popup menu Repeat for any other Biosearch dyes Click the Show Analysis button and your plate layout will turn entirely black Click Run and the instrument will begin collecting the calibration data When the instrument has completed the data collection select the four wells in the plate layout that correspond to a calibration dye Select Calibrate from the Instrument menu and then select Extract Pure Dye Click OK on the following confirmation screen You will now be presented with a screen showing the emission spectra of each of your four replicate wells as well as an emission spectrum representing the average of these replicates If any of the spectra deviate from the rest of the group remove these from the average and then click OK Repeat for the other dyes you are calibrating simultaneously Quit the application Calibration has been c
11. he Master Pure Dye file elsewhere on the computer for future use Spectral Calibration FINAL 092305 doc BIOSEARCH es TECHNOLOGIES E i Chemistry jor Genomics and Proteomics Bio RAD LABORATORIES CYCLER Following is a general procedure for calibrating Biosearch dyes on the Bio Rad iCycler For further details please refer to the iCycler iQ Real Time Detection System Resource Guide that describes the calibration procedure on pages 55 58 and the following table for recommended concentrations Calibration Concentration on the Approximate of Calibrations CAL Fluor Gold 540 CAL Fluor Orange 560 CAL Fluor Red 590 CAL Fluor Red 610 CAL Fluor Red 635 Quasar 570 Quasar 670 1 Calibration data for the iCycler is saved to the file RME ini located in the directory C Program Files Bio Rad iCycler Ini By opening this file in a word processor you can view the spectral profile for each of the dyes that are currently calibrated More information on this file is available in Appendix I of the Resource Guide Before performing calibration of any Biosearch dyes it is important to make a backup copy of this file with an appropriate name such as Original RME ini PAM CAL Fluor Gold 540 CAL Fluor Orange 560 CAL Fluor Red 590 CALFluorRed610 CALFluorRed635 Ouasar570 O O Quasare70 O po 2 To ensure that the iCycler collects spectral data on all relevant channels It is necessary to anticipate which fluorescen
12. he plate and place it into the instrument 2 Start the Opticon Monitor 2 application and select Dye Calibration from the Tools drop down menu You will be presented with a Dye Calibration Wizard In the Plate type field choose the appropriate reaction plate white or clear Within the Dye name field type a name for the Biosearch dye that you wish to calibrate eg CALgold Click Next 3 Click Go and the instrument will begin acquiring the data Shortly the instrument will pause and prompt you to rotate the plate 180 After doing so click Go 4 Once the measurements have finished inspect the results A problem with the plate will be indicated by anomalous values for one or more wells If this is observed discard the plate and start over Otherwise click the Next button Note the general signal intensity of the blank plate in each of the detection channels 5 Remove the empty plate from the instrument and discard the optical cover or caps Dilute the Biosearch Calibration dye in 1X PCR buffer to the necessary working concentration refer to Table 1 and a volume of 5 mL Mix well 6 Dispense 50 uL of this calibration solution into all 96 wells of the same reaction plate that was just removed from the instrument Seal the plate with a new optical adhesive cover position a compression pad on top and place it back into the instrument Click Go Shortly the instrument will pause and prompt you to rotate the plate 180 Aft
13. in setting was too high for your amplification on that channel If endpoint fluorescence is important for your data analysis then it will be necessary to recalibrate that channel using a lower concentration that elicits a reduced gain setting Repeat this calibration procedure step 6 gt step 17 for the remaining 12 reactions sites on the instrument Once again use care to not scratch the tubes or introduce bubbles It is also important to keep all dye names and settings the same as with your previous calibration Once these are complete you are ready to run your multiplexed assay on the calibrated reaction sites Spectral Calibration FINAL 092305 doc 4 Page 11 of 12 TECHNOLOGIES T i ine PRITY for Generic and Proteomics MJ RESEARCH OPTICON 2 Following is a general procedure for calibrating Biosearch custom dyes on the MJ Research Opticon 2 For further details please refer to the DNA Engine Opticon 2 System Troubleshooting Calibration pages 10 2 through 10 6 that describe the calibration procedure and the following table recommended concentrations Calibration Concentration on the Approximate of Calibrations 3 PAM 80M 1 The Opticon 2 first measures an empty reaction plate to subtract blank values from the subsequent calibration measurements Obtain an empty plate of the same type you will use for future real time PCR reactions and seal it using an optical adhesive cover Place a compression pad on top of t
14. martCycler Operator Manual Appendix B and the application note User Defined Optical Calibration This document also outlines the procedure for determining the optimum dye concentration for calibration CUSTOMER SERVICE AND TECHNICAL SUPPORT If you require additional information or technical assistance please feel free to email our Technical Support group at techsupport biosearchtech com For immediate assistance our knowledgeable staff is available for telephone consultation from 7 30 AM to 5 00 PM Monday through Friday Pacific Coast time Please contact us at 1 800 GENOME 1 436 6631 U S Canada only 1 415 883 8400 telephone 1 415 883 8488 fax Spectral Calibration FINAL 092305 doc BIOSEARCH eae TECHNOLOGIES T z 1 Cine PRITY for Genomics and Proteomics INSTRUMENT SPECIFIC CALIBRATION INSTRUCTIONS ABI PRISM 7700 SEQUENCE DETECTION SYSTEM Following is a general procedure for calibrating Biosearch dyes on the ABI 7700 and a table listing the recommended dye concentrations For further details please refer to the ABI User Bulletin 4 Generating New Spectral Components as well as ABI Prism 7700 User s Manual that describes the calibration procedure on pages 3 9 to 3 12 Calibration Dve Calibration Concentration on the Approximate of Calibrations y Prism 7700 possible 200 nM 125 FAM 125 100 incompatible CAL Fluor Red 635 Incompatible Incompatible Quasar 570 Incompatible Incompatible Quasar 670 I
15. ncompatible Incompatible 1 Prior to running spectral calibration with your Biosearch dye you will need to generate a background component file This procedure allows the instrument to measure the background signal of each well and to compensate for any residual fluorescence contaminating the heating block 2 Dispense 50 uL of deionized water into each well of an optical PCR reaction plate Seal the plate with an optical adhesive cover and place a compression pad on top of the plate Position the plate into the instrument and tighten the lid 3 Start the SDS software application and close the plate layout that opens automatically Select New Plate from the File menu designate the plate type as Background and click OK Click Show Analysis and the wells depicted in the plate layout will turn black Click Run 4 After the run has completed select Calibrate from the Instrument menu Within the Calibrate submenu select Extract Background Component You will then be prompted to quit the application The background data has been stored regardless of whether you choose to save or discard the file containing the plate setup You are now ready to prepare your calibration plate containing the Biosearch dye 5 Dilute the Biosearch Calibration dye s in 1X PCR buffer to the necessary working concentration according to the above table and a volume of 250 uL Mix well 6 For each calibration dye dispense 50 uL of this dye solution into each
16. of 4 wells in an optical PCR reaction plate This will leave 50 uL excess which can be discarded Seal the plate with an optical adhesive cover and place a compression pad on top of the plate Position the plate into the instrument and tighten the lid 7 Open the SDS software application close the existing plate layout and select New Plate from the File menu Designate plate type as Pure Spectra and click OK 8 Within the Sample Type field select Sample Type Setup You will need to create a new entry for each Biosearch dye by clicking Add A blank entry will be added to the bottom of the list Within the Name field for this entry tyoe Pure Dye exactly as spelled Spectral Calibration FINAL 092305 doc lt BIOSEARCH Page 5 of 12 SS TECHNOLOGIES T z 1 R t PEMTGITY for Genomics and Proteomics 9 xr xr SNr xr xr Within the Acronym field type a short name for the new dye name using the following conventions the letter Z followed by the acronym that you wish to use For example ZORG could be used to describe CAL Fluor Orange 560 Note This naming convention is important for the following reasons the ABI 7700 allows you to calibrate for an unlimited number of custom dyes but a software bug requires users to maintain FAM TAMRA and ROX within the first seven entries alphabetically This list of calibrated dyes can be viewed by selecting Edit Pure Dye within the Instrument gt Calibrate menus Adding a Z in
17. ompleted and a file titled Pure Dye has now been updated to include the new dye s in addition to the previous standard dyes This file is stored in the following directory System Folder Preferences SDS Spectra Components Importantly backup this Pure Dye file with an appropriate name such as Master Pure Dye The Pure Dye file will serve as your dye palette when running future experiments and can be viewed by selecting Edit Pure Dye within the Instrument Calibrate menus Just prior to performing a real time PCR experiment with the Biosearch dye s you will need to delete dye entries that will remain unused during the experiment using the Edit Pure Dye window IMPORTANT NOTE filtering out unused dyes places the Biosearch dye within the first seven entries enabling the SDS software to recognize it This will permanently modify the Pure Dye file which is why the backup described in step 16 is essential IF THE PURE DYE FILE HAS NOT BEEN BACKED UP DELETED ENTRIES CAN ONLY BE REGAINED THROUGH RECALIBRATION Due to the software limitation described in step 9 above the entries for FAM TAMRA and ROX should NEVER be removed or the application will not open properly Once you have completed real time PCR with your Biosearch custom dye you must restore the dye entries that were removed Quit the SDS application and replace the current Pure Dye file with the Master Pure Dye backup file making the appropriate name changes Always keep a copy of t
18. on dye in your chosen PCR buffer Store at 20 C until you are ready to dilute to the working concentration and perform spectral calibration STORAGE AND HANDLING Calibration dyes should be subjected to a minimum number of freeze thaw cycles Therefore we recommend that you prepare microvials each having sufficient material for one dye calibration and store them frozen at 20 C or 80 C Calibration dyes can be stored frozen in solution for over one year PROTECTION FROM PHOTOBLEACHING To ensure optimum activity and to safeguard maximum performance lifetime calibration dyes should always be protected from light to avoid photobleaching OPTIONAL REACTION PLATE CENTRIFUGATION When dispensing calibration dyes into a reaction plate care should be taken to ensure that bubbles are not introduced and that solution hasn t collected on the side of a well Briefly centrifuging the plate lt 1500xg for 5 seconds can correct these problems but otherwise is not required CAL Fluor Red 610 RD 5082 95 00 Spectral Calibration FINAL 092305 doc Page 3 of 12 BIOSEARCH SS TECHNOLOGIES oa i Chemistry jor Genomics and Proteomics STORING AND SAVING CALIBRATION PLATES FOR FUTURE USE After successfully calibrating your real time PCR instrument the reaction plate containing your calibration dye can be frozen at 20 C and re used in the future for subsequent calibrations Most instruments recommend periodic recalibration Cons
19. os 4 683 195 4 683 202 and 4 965 188 and by issued and pending counterparts outside the U S These patents are owned by RocheMolecular Systems Inc and have been sub licensed by PE Corporation in certain fields Depending on your specific application you may need a license from Roche or PE to practice PCR Additional information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems 1145 Atlantic Avenue Alameda CA 94501 or Applied Biosystems business group of the Applera Corporation 850 Lincoln Centre Drive Foster City CA 94404 In addition the 5 nuclease assay and other homogeneous amplification methods used in connection with the PCR process may be covered by U S Patents 5 210 015 and 5 487 972 owned by Roche Molecular Systems Inc and by U S Patent 5 538 848 owned by The Perkin Elmer Corporation The purchase of Biosearch Technologies products does not either expressly or by implication provide a license to use this or other patented technology Licensing information can be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 or the Licensing Department at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda CA 94501 Other Disclaimers TaqMan is a registered trademark of Roche Molecular Systems Inc Alameda CA Prism is a registered trademark of Applied Biosystems Foster City
20. propriate name such as Current RME ini You are now prepared to run real time PCR using your custom Biosearch dye on the iCycler In setting up the plate layouts for these future reactions remember to use the standard dye entry s upon which your Biosearch dyes are calibrated Spectral Calibration FINAL 092305 doc Page 8 of 12 BIOSEARCH TECHNOLOGIES oa i Chemistry for Genomics and Proteomic a CEPHEID SMARTCYCLER II Following is a general procedure for calibrating Biosearch dyes on the Cepheid SmartCycler II and a table listing the recommended dye concentrations For further details please refer to the SmartCycler Application Note User Defined Optical Calibration as well as the SmartCycler Operator Manual that describes the calibration procedure in Appendix B Calibration Concentration on the Appr Cee of leo Calibration Dye SmartCvcler II possible single processing y block 25 uL volumes AM 0M Note there is considerable variation in the sensitivity of fluorescence detection from one I CORE reaction site to the next While we have strived to determine a universal calibration concentration for each dye it is possible that these recommendations might produce too low a fluorescent signal to successfully calibrate one or more sites on your instrument Alternately the concentration might be too high resulting in a low gain setting and future amplifications that are poorly detected We recommend fine tuning these suggested
21. t dyes you might use together on a reaction plate and calibrate for all of those simultaneously in one run For example if you multiplex a FAM CAL Fluor Orange 560 and CAL Fluor Red 610 assay but had calibrated for each of these dyes on separate days an error message might result stating Response Matrix element not found in RME database Recalibration is then necessary 3 Dilute each Biosearch Calibration dye in 1X PCR buffer to the working concentration of 300 nM and a volume of 550 uL Mix well 4 For each dye dispense 50 uL of the Biosearch calibration solution into each of 10 wells in an optical PCR reaction plate This will leave you with a 50 uL excess for each calibration solution which you can discard Seal the plate with an optical adhesive cover and then save it until you are prompted to place it into the iCycler 5 Start the iCycler software application In the Library mode check to make sure that you are in the View Protocol pane and select the PureDyeCalibration tmo themal cycling protocol 6 Switch to the View Plate Setup pane and select the Pure dye pts plate setup Click the button Edit this Plate Setup and you will be transferred to the Workshop mode of the application 7 Clear the existing well labels by selecting the Erase function and clicking the top left corner of the plate layout 8 Next select the Pure Dye button and label the wells on the plate layout that correspond to the wells in the reaction pl
22. the Gain Normalized Signal GNS Spectral Calibration FINAL 092305 doc BIOSEARCH Page 10 of 12 SS TECHNOLOGIES T z 1 A t PEMTGITY for Genomics and Proteomics 16 17 18 19 20 or the Signal to Background ratio S B is too low Both of these errors indicate that calibration needs to be repeated with the concentration of one of the dyes boosted to a higher value The verification procedure will require the same set of tubes to be placed into the instrument again but it is highly recommended that you shuffle their placement so that the same exact tube will not be placed back into the same exact reaction site Before beginning verification we recommend taking this moment to spin them down in the minifuge so that any bubbles introduced during the previous steps can be removed Follow the prompts during the verification procedure Upon completion you will be prompted to restart the SmartCycler Il application After the application has finished starting up we recommend returning to the Optical Calibration window and clicking the Report button to view the results of the last calibration procedure Make a note of the Gains associated with each channel to understand the intensity of your calibration dyes within the instrument s range of detection The gains run from 033 If you ever witness amplifications that abruptly plateau in your future PCR reactions this is a sign that the optics have been saturated and that the ga
23. ult your instruments manual for the recommended frequency of recalibration CALIBRATION INFORMATION FOR UNSUPPORTED INSTRUMENTS For certain real time PCR instruments we are unable to provide specific calibration instructions or recommended concentrations If your instrument is one of the following please refer to the alternate sources of information for the appropriate protocols or contact your instrument manufacturer directly ABI Prism 7000 Sequence Detection System Please refer to the Sequence Detection System User Guide Chapter 8 Pure Dye Assay Pure Dyes pages 8 17 to 8 22 ABI Prism 7300 Sequence Detection System Please refer to the document Installation and Maintenance Applied Biosystems 7300 7500 Real Time PCR System pages 57 72 This document also outlines the procedure for determining the optimum dye concentration for calibration ABI Prism 7500 Sequence Detection System Please refer to the document Installation and Maintenance Applied Biosystems 7300 7500 Real Time PCR System pages 57 72 This document also outlines the procedure for determining the optimum dye concentration for calibration ABI Prism 7900 Sequence Detection System Please refer to the document ABI Prism 7900HT Sequence Detection System User Guide Chapter 7 Performing a Pure Dye Run pages 7 17 to 7 23 This document also outlines the procedure for determining the optimum dye concentration for calibration SmartCycler Please refer to the S
24. verwrite the previous settings for that entry which is why the backup described in step 1 is essential If the need ever arises to use the original dye that was replaced by the Biosearch dye the backup RME ini file can be substituted for the modified one Check the box of the standard dye entry for which you will calibrate the Biosearch dye You will be immediately prompted to select a color representing this dye On the plate layout select the wells that you designated as Pure Dye and they will be filled in with the color of this entry Repeat for any additional Biosearch dyes that you will be calibrating simultaneously After you have designated the location of the calibration dye on your plate layout click Save this plate setup and finally Run with selected protocol The screen will switch to the Run Prep pane and at this point you should place an external well factor plate into the instrument Preparation of an external well factor plate is described on page 54 of the Resource Guide Select Begin Run and the instrument will prompt you to enter a name for the optical data file that will be generated The iCycler will first collect optical data for the well factors When the iCycler pauses open the instrument remove the well factor plate and replace it with the reaction plate containing your calibration dyes Click Continue Running Protocol When calibration has completed you need to make a backup of the now modified RME ini file with an ap
Download Pdf Manuals
Related Search