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Soft And Handling

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Soft And Handling

  1. 1. Creating a Run
  2. 2. 1. Click the Create Run icon 2. Enter a unique run name 3. Select dye set 4. Click the Add/Remove sites button
  3. 3. 5. Select the protocol(s)-select a different protocol for each site. 6. Select sites – use ctrl and shift to select multiple sites 7. Click right-pointing arrow to transfer protocol and sites to the selections column. 8. Verify that selection is correct then click OK.
  4. 4. Defining a Protocol
  5. 5. 2. Click New Protocol button 1. Click Define Protocols icon 3. Enter a unique protocol name and click OK. 4. Enter thermal cycling protocol. 5. Click Save Protocol
  6. 6. Advance to Next Stage feature – the protocol will automatically advance to the next PCR stage after the threshold crossing.
  7. 7. Defining a Graph
  8. 8. 1. Click Define Graphs icon 2. Click New Graph button 3. Enter a unique graph name and click OK. 4. Enter graph definition 5. Click Save Graph
  9. 9. Viewing Results
  10. 10. Compare two runs Customize Views list View temperature and optical data in real time Customize data analysis
  11. 11. Viewing Results Table
  12. 12. <ul><li>Setup standard curves </li></ul><ul><li>Sample IDs </li></ul><ul><li>View C t values </li></ul><ul><li>View Melt Temperatures </li></ul>
  13. 13. Background subtraction
  14. 14. Background subtraction <ul><li>The background is subtracted initially at cycle 13 </li></ul><ul><ul><ul><li>Background Min cycle is 5 </li></ul></ul></ul><ul><ul><ul><li>At least 5 cycles are required for this calculation </li></ul></ul></ul><ul><ul><ul><li>To avoid using fluorescence data derived from amplified DNA, the 4 most recent cycles are not used </li></ul></ul></ul><ul><li>The background subtraction calculation stops 4 cycles before the threshold crossing. </li></ul>
  15. 15. Background subtraction Background subtraction and drift correction started at cycle 5 and stopped at cycle 28.39.
  16. 16. Background subtraction Appearance of negative drift because positive slope of the growth curve was included in the background subtraction and drift correction calculation. Threshold set too high
  17. 17. Smart Cycler Menus
  18. 18. Smart Cycler Menus <ul><li>Administration </li></ul><ul><ul><li>Login/Logout </li></ul></ul><ul><ul><li>Search by run name or specimen </li></ul></ul><ul><ul><li>Limit access to runs, protocols and graphs </li></ul></ul><ul><li>Customization </li></ul><ul><ul><li>Analysis Settings </li></ul></ul><ul><ul><li>Export data (jpeg and excel files) </li></ul></ul><ul><ul><li>Melt analysis </li></ul></ul><ul><li>Automatic backup of database </li></ul>
  19. 19. Melt Settings
  20. 20. Control <ul><li>Internal Control </li></ul><ul><ul><li>Used to validate an assay confirming that the reagents are functional. </li></ul></ul><ul><li>Quantitative Internal Control </li></ul><ul><ul><li>Used to correct for differences in assay performance due to normal site-to-site and run-to-run variations. </li></ul></ul>
  21. 21. Quantification Strategy <ul><li>Absolute </li></ul><ul><ul><li>requires standard whose concentration is known absolutely </li></ul></ul><ul><ul><li>uses standard curve </li></ul></ul><ul><ul><li>unnecessary for most studies </li></ul></ul><ul><li>Relative </li></ul><ul><ul><li>requires reference gene </li></ul></ul><ul><ul><li>uses normalization </li></ul></ul><ul><ul><li>Assumption that control doesn’t vary under the experimental conditions. </li></ul></ul>
  22. 22. Absolute Quantification <ul><li>The fact that this method relies on a set of knows is the reason it cannot be “absolute”. No matter what the source or how carefully it is measured, there is no way to know exactly how much or how many copies of a known template truly exists in a given well of a known sample. </li></ul><ul><li>Gene quantification using Real Time Quantitative PCR : an emerging technology hits the mainstream. David G. Ginzinger, Experimental Hematology 30 (2002) 503-512. </li></ul>
  23. 23. Relative Quantification <ul><li>Current methods to determine exact numbers of molecules overcome the determination of the amplification rate by assuming identical amplification rates for a target DNA sequence and a standard of known quantity introduced into the experiment design, so that only the ratio of amplified products need be determined. Violations of the hypothesis of identical amplification rates for two sequences will result in a systematic bias in the experiment results that underestimates or overestimates the initial copy numbers. </li></ul><ul><li>Statistical Estimations of PCR Amplification Rates. Jean Peccoud and Christine Jacob. </li></ul>
  24. 24. Quantification 10 1 10 2 10 3 10 4 10 5 Unknown Sample Threshold
  25. 25. Quantification
  26. 26. SYBR ® Green – Standard Curve (Roche FastStart DNA SYBR Green kit; each dilution run in triplicate)
  27. 27. Setting up a Standard Curve
  28. 28. <ul><li>Select Sample Type </li></ul><ul><li>Enter standard concentration </li></ul><ul><li>Click Update Analysis button </li></ul><ul><li>View Standard Curve graph </li></ul>
  29. 29. Importing a Standard Curve
  30. 30. Importing a Standard Curve
  31. 31. Importing a Standard Curve Imported standard curve is highlighted in yellow.
  32. 32. Saving an Imported Standard Curve
  33. 33. Quantitation of Unknowns
  34. 34. Maintenance Screen
  35. 35. Smart Cycler Menus User Administration Login/Logout
  36. 36. Login
  37. 37. Smart Cycler Menus Run and Specimen logs allow the user to search by run name or specimen name.
  38. 38. Run log
  39. 39. Run Report
  40. 40. Specimen report
  41. 41. Specimen report
  42. 42. Specimen report
  43. 43. User Administration <ul><li>User Administration can be used to: </li></ul><ul><li>Sort runs by user name </li></ul><ul><li>Identify protocols and graphs by user name </li></ul><ul><li>Limit access to your runs, protocols and graphs </li></ul>
  44. 44. User Administration tamlyn tamlyn **** **** Enter a User Name and Password
  45. 45. Configure User Select User name User Rights.
  46. 46. Setup Menu- System Defaults <ul><li>Customize default Analysis Settings </li></ul><ul><li>Automatic backup of database </li></ul><ul><li>Customize exported data and set automatic export </li></ul><ul><li>Customize melt analysis </li></ul><ul><li>Limit user access to their own runs, protocols and graphs </li></ul>
  47. 47. General
  48. 48. Analysis Setting
  49. 49. Automatic Backup
  50. 50. Default Export Settings
  51. 51. Default Export Settings
  52. 52. Default Export Settings
  53. 53. Export graph data
  54. 54. Export graph data
  55. 55. Melt Settings
  56. 57. Melt Settings
  57. 58. Melt Settings
  58. 59. Melt Settings
  59. 60. Melt Settings
  60. 61. Melt Settings
  61. 62. Melt Settings
  62. 63. Access Option
  63. 64. Smart Cycler Menus
  64. 65. Smart Cycler Menus
  65. 67. <ul><li>At Calibration: </li></ul><ul><ul><li>Measure raw values with buffer </li></ul></ul><ul><ul><li>Measure raw values with each pure dye. </li></ul></ul><ul><ul><li>Determine ‘net’ dye signals for each pure dye and construct signal matrix. </li></ul></ul><ul><ul><li>Invert matrix to determine ‘calibration’ matrix </li></ul></ul><ul><li>During a run </li></ul><ul><ul><li>Measure raw data of unknown mix </li></ul></ul><ul><ul><li>Subtract off stored ‘buffer’ values </li></ul></ul><ul><ul><li>Multiply by calibration matrix to obtain ‘deconvolved’ calibrated signals </li></ul></ul>Optical Calibration Issues
  66. 68. Optical Calibration Issues <ul><li>The emission spectra of fluorescent dyes is quite broad. </li></ul><ul><li>A pure dye produces signal in more than one optical channel. For example, TET produces raw signal in Ch1, Ch 2 and Ch 3: </li></ul>
  67. 69. Optical Calibration Issues (cont) <ul><li>After calibration, we want each pure dye to give a signal </li></ul><ul><li>of 1000 fluorescent units only in its appropriate channel: </li></ul>
  68. 70. Optical Calibration Issues (cont) <ul><li>In reality, we see some ‘dye crosstalk’ between the channels, so we might see data like below after calibration: </li></ul>
  69. 71. Signal Analysis - Version 1 and 2
  70. 72. Help Menus
  71. 73. Help Menus
  72. 74. Software Diagnostic
  73. 75. Software Diagnostic cmartcycleroolsyclerdiagnostics Tools used by technicians/engineers to perform functional testing and trouble-shooting of the smartcycler
  74. 76. Software Diagnostic Command Menu Site(s) activation table Cycler Diagnostic Rev.
  75. 77. Software Diagnostic GetDeviceSN : Reports the serial number of the instrument, backplane, the CPU board, the instrument model number and bootcode revision number
  76. 78. Software Diagnostic
  77. 79. Software Diagnostic
  78. 80. Troubleshooting examples
  79. 81. <ul><li>Obtain the following required information: </li></ul><ul><li>Serial number of Computer </li></ul><ul><li>Serial number of Smart Cycler </li></ul><ul><li>Additional info (change form original) </li></ul><ul><li>Computer Model and OP </li></ul><ul><li>Version of Smart Cycler Software </li></ul><ul><li>Version of the Anti-Virus software </li></ul>Troubleshooting Computer Problems
  80. 82. <ul><li>Problem description : </li></ul><ul><li>Was the software open when the problem occurred? </li></ul><ul><li>Was a run in progress when the problem occurred? </li></ul><ul><li>What applications were open when the problem occurred? </li></ul><ul><li>Is the computer networked? </li></ul>Troubleshooting Computer Problems
  81. 83. <ul><li>General Computer Information: </li></ul><ul><li>SSQL </li></ul><ul><li>USB devices </li></ul><ul><li>Printers </li></ul><ul><li>Windows 2000 </li></ul><ul><ul><li>Login </li></ul></ul><ul><ul><li>Set all power settings to Never </li></ul></ul><ul><li>Additional Software </li></ul><ul><li>The Smart Cycler software license allows the user to load the software on1 additional computer for data analysis </li></ul>Troubleshooting Computer Problems
  82. 84. <ul><li>Most Frequent : </li></ul><ul><li>Loss of communication: </li></ul><ul><ul><li>Using more than one block? </li></ul></ul><ul><ul><li>USB cables </li></ul></ul><ul><ul><li>Additional software/hardware </li></ul></ul><ul><ul><li>Networking/Internet </li></ul></ul><ul><ul><li>Norton Anti-virus </li></ul></ul><ul><ul><li>Power questions </li></ul></ul><ul><li>The computer is running slow </li></ul><ul><li>The Database Cannot be Connected </li></ul><ul><li>Windows 2000 </li></ul>Troubleshooting Computer Problems
  83. 85. Frequently Asked Questions
  84. 86. <ul><li>Acceptable temperature range : 40  C to 98  C </li></ul><ul><li>Can I backup my data? Yes, individual runs can be archived and retrieved or the entire database can be backed up </li></ul><ul><li>What is the capacity of the database? 1.9GB </li></ul><ul><li>Can I use VIC on the Smart Cycler? Smart Cycler II system must be recalibrated with the user-defined Optical Calibration </li></ul><ul><li>Can I fill the 100ul tube with 50ul? No </li></ul><ul><li>Can I use 100ul and 25ul together in a single run? No </li></ul><ul><li>And more … </li></ul>General Software Questions
  85. 87. Rules 1.White coat must be worn at all times in the laboratory. Coats are stored in the laboratory. Coats are removed before leaving the laboratory. 2.Never ingest any chemical from lab and, if a chemical is spilled on your hands, wash them immediately 3.You must ALWAYS thoroughly wash and scrub your hands before you leave the laboratory. 4.Follow the specific safety rules for each hazardous chemical. Each hazardous chemical will be given to you with specific instructions for its handling. These vary from chemical to chemical depending on the hazard each presents. Follow these instructions exactly and ask for assistance if you are unsure about how to proceed.
  86. 88. How remove gloves Wash your hands. finish turn the first glove inside out and discard it … turn completly the second glove inside out and discard it 6 5 4 With fingers wrapped in glove turned inside out take the second glove off… Remove glove till appearance. Seize glove few centimeters from glove rims. 3 2 1
  87. 89. Recommendations relating to hand washing Use towel to turn off water and discard in open bin Dry hands with disposable hand towel. Thoroughly rinse. 6 5 4 Rub hands together for 30 secondes, work all surfaces, insist on palms. Collect one dose of liquid soap. Roll up sleeves first and wet hands. 3 2 1

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