Soft And Handling
Upcoming SlideShare
Loading in...5
×
 

Soft And Handling

on

  • 559 views

 

Statistics

Views

Total Views
559
Views on SlideShare
559
Embed Views
0

Actions

Likes
0
Downloads
9
Comments
0

0 Embeds 0

No embeds

Accessibility

Categories

Upload Details

Uploaded via as Microsoft PowerPoint

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

Soft And Handling Soft And Handling Presentation Transcript

  • Creating a Run
  • 1. Click the Create Run icon 2. Enter a unique run name 3. Select dye set 4. Click the Add/Remove sites button
  • 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.
  • Defining a Protocol
  • 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
  • Advance to Next Stage feature – the protocol will automatically advance to the next PCR stage after the threshold crossing.
  • Defining a Graph
  • 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
  • Viewing Results
  • Compare two runs Customize Views list View temperature and optical data in real time Customize data analysis
  • Viewing Results Table
    • Setup standard curves
    • Sample IDs
    • View C t values
    • View Melt Temperatures
  • Background subtraction
  • Background subtraction
    • The background is subtracted initially at cycle 13
        • Background Min cycle is 5
        • At least 5 cycles are required for this calculation
        • To avoid using fluorescence data derived from amplified DNA, the 4 most recent cycles are not used
    • The background subtraction calculation stops 4 cycles before the threshold crossing.
  • Background subtraction Background subtraction and drift correction started at cycle 5 and stopped at cycle 28.39.
  • 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
  • Smart Cycler Menus
  • Smart Cycler Menus
    • Administration
      • Login/Logout
      • Search by run name or specimen
      • Limit access to runs, protocols and graphs
    • Customization
      • Analysis Settings
      • Export data (jpeg and excel files)
      • Melt analysis
    • Automatic backup of database
  • Melt Settings
  • Control
    • Internal Control
      • Used to validate an assay confirming that the reagents are functional.
    • Quantitative Internal Control
      • Used to correct for differences in assay performance due to normal site-to-site and run-to-run variations.
  • Quantification Strategy
    • Absolute
      • requires standard whose concentration is known absolutely
      • uses standard curve
      • unnecessary for most studies
    • Relative
      • requires reference gene
      • uses normalization
      • Assumption that control doesn’t vary under the experimental conditions.
  • Absolute Quantification
    • 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.
    • Gene quantification using Real Time Quantitative PCR : an emerging technology hits the mainstream. David G. Ginzinger, Experimental Hematology 30 (2002) 503-512.
  • Relative Quantification
    • 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.
    • Statistical Estimations of PCR Amplification Rates. Jean Peccoud and Christine Jacob.
  • Quantification 10 1 10 2 10 3 10 4 10 5 Unknown Sample Threshold
  • Quantification
  • SYBR ® Green – Standard Curve (Roche FastStart DNA SYBR Green kit; each dilution run in triplicate)
  • Setting up a Standard Curve
    • Select Sample Type
    • Enter standard concentration
    • Click Update Analysis button
    • View Standard Curve graph
  • Importing a Standard Curve
  • Importing a Standard Curve
  • Importing a Standard Curve Imported standard curve is highlighted in yellow.
  • Saving an Imported Standard Curve
  • Quantitation of Unknowns
  • Maintenance Screen
  • Smart Cycler Menus User Administration Login/Logout
  • Login
  • Smart Cycler Menus Run and Specimen logs allow the user to search by run name or specimen name.
  • Run log
  • Run Report
  • Specimen report
  • Specimen report
  • Specimen report
  • User Administration
    • User Administration can be used to:
    • Sort runs by user name
    • Identify protocols and graphs by user name
    • Limit access to your runs, protocols and graphs
  • User Administration tamlyn tamlyn **** **** Enter a User Name and Password
  • Configure User Select User name User Rights.
  • Setup Menu- System Defaults
    • Customize default Analysis Settings
    • Automatic backup of database
    • Customize exported data and set automatic export
    • Customize melt analysis
    • Limit user access to their own runs, protocols and graphs
  • General
  • Analysis Setting
  • Automatic Backup
  • Default Export Settings
  • Default Export Settings
  • Default Export Settings
  • Export graph data
  • Export graph data
  • Melt Settings
  •  
  • Melt Settings
  • Melt Settings
  • Melt Settings
  • Melt Settings
  • Melt Settings
  • Melt Settings
  • Access Option
  • Smart Cycler Menus
  • Smart Cycler Menus
  •  
    • At Calibration:
      • Measure raw values with buffer
      • Measure raw values with each pure dye.
      • Determine ‘net’ dye signals for each pure dye and construct signal matrix.
      • Invert matrix to determine ‘calibration’ matrix
    • During a run
      • Measure raw data of unknown mix
      • Subtract off stored ‘buffer’ values
      • Multiply by calibration matrix to obtain ‘deconvolved’ calibrated signals
    Optical Calibration Issues
  • Optical Calibration Issues
    • The emission spectra of fluorescent dyes is quite broad.
    • A pure dye produces signal in more than one optical channel. For example, TET produces raw signal in Ch1, Ch 2 and Ch 3:
  • Optical Calibration Issues (cont)
    • After calibration, we want each pure dye to give a signal
    • of 1000 fluorescent units only in its appropriate channel:
  • Optical Calibration Issues (cont)
    • In reality, we see some ‘dye crosstalk’ between the channels, so we might see data like below after calibration:
  • Signal Analysis - Version 1 and 2
  • Help Menus
  • Help Menus
  • Software Diagnostic
  • Software Diagnostic csmartcycler oolscyclerdiagnostics Tools used by technicians/engineers to perform functional testing and trouble-shooting of the smartcycler
  • Software Diagnostic Command Menu Site(s) activation table Cycler Diagnostic Rev.
  • Software Diagnostic GetDeviceSN : Reports the serial number of the instrument, backplane, the CPU board, the instrument model number and bootcode revision number
  • Software Diagnostic
  • Software Diagnostic
  • Troubleshooting examples
    • Obtain the following required information:
    • Serial number of Computer
    • Serial number of Smart Cycler
    • Additional info (change form original)
    • Computer Model and OP
    • Version of Smart Cycler Software
    • Version of the Anti-Virus software
    Troubleshooting Computer Problems
    • Problem description :
    • Was the software open when the problem occurred?
    • Was a run in progress when the problem occurred?
    • What applications were open when the problem occurred?
    • Is the computer networked?
    Troubleshooting Computer Problems
    • General Computer Information:
    • SSQL
    • USB devices
    • Printers
    • Windows 2000
      • Login
      • Set all power settings to Never
    • Additional Software
    • The Smart Cycler software license allows the user to load the software on1 additional computer for data analysis
    Troubleshooting Computer Problems
    • Most Frequent :
    • Loss of communication:
      • Using more than one block?
      • USB cables
      • Additional software/hardware
      • Networking/Internet
      • Norton Anti-virus
      • Power questions
    • The computer is running slow
    • The Database Cannot be Connected
    • Windows 2000
    Troubleshooting Computer Problems
  • Frequently Asked Questions
    • Acceptable temperature range : 40  C to 98  C
    • Can I backup my data? Yes, individual runs can be archived and retrieved or the entire database can be backed up
    • What is the capacity of the database? 1.9GB
    • Can I use VIC on the Smart Cycler? Smart Cycler II system must be recalibrated with the user-defined Optical Calibration
    • Can I fill the 100ul tube with 50ul? No
    • Can I use 100ul and 25ul together in a single run? No
    • And more …
    General Software Questions
  • 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.
  • 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
  • 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