Bio-Rad Firmware & Software Updates - Manual (Page 70)

MiniOpticon Instruction Manual
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Baseline Settings
The software automatically sets the baseline individually for each well. Select the Baseline
Setting to determine the method of baseline subtraction for all fluorescence traces. Select
Settings > Baseline Setting to choose one of these three options:
• No Baseline Subtraction. The software displays the data as relative fluorescence
traces. Some analysis is not possible in this analysis mode, and therefore the software
does not display the Gene Expression, End Point, and Allelic Discrimination tabs
• Baseline Subtracted. The software displays the data as baseline subtracted traces for
each fluorophore in a well. The software must baseline subtract the data to determine
quantification cycles, construct standard curves, and determine the concentration of
unknown samples. To generate a baseline subtracted trace, the software fits the best
straight line through the recorded fluorescence of each well during the baseline cycles,
and then subtracts the best fit data from the background subtracted data at each cycle
• Baseline Subtracted Curve Fit. The software displays the data as baseline subtracted
traces, and the software smoothes the baseline subtracted curve using a centered mean
filter. This process is performed so that each C
q
is left invariant
Along with the options above, the following can also be selected:
• Apply Fluorescent Drift Correction. For wells that have abnormally drifting RFU values
during the initial few cycles of a run the software derives an estimated baseline from
adjacent wells for which a horizontal baseline was successfully generated
Adjusting the Baseline
Once wells for analysis have been selected, check the baseline settings in these wells. Open
the Baseline Thresholds window (Figure 40) to change the default baseline for selected wells.
To open this window:
1. Select a single fluorophore in the Quantification tab (Figure 37) by clicking the boxes next
to the fluorophore name located under the Amplification chart.
2. Select Settings > Baseline Threshold to open the Baseline Threshold window.
To adjust the begin and end baseline cycle for each well:
1. In the Baseline Cycles pane, select one or more wells by clicking the row number,
clicking the top left corner to select all wells, holding down the Control key to select
multiple individual wells, or holding down the shift key to select multiple wells in a row.
2. Adjust the Baseline Begin cycle and Baseline End cycle for all selected wells or change
the Begin and End cycle number at the bottom of the spreadsheet (Figure 40).
3. To revert the settings back to the last saved values, click Reset All User Defined
Values.
4. Click OK to confirm any changes and close the window.
Analysis Mode
Data can be analyzed and displayed grouped by either fluorophore or target name. To choose
the data analysis mode, select Settings > Analysis Mode or make a selection from the
Analysis Mode drop down menu in the toolbar.