BIO RAD CFX96 User Manual

CFX96 and CFX384 Systems Manual
95
• Change a call manually by highlighting a row in the spreadsheet, and then selecting
an option in the Call Selected Alleles list (including Allele 1, Allele 2, Heterozygote,
None, Unknown, Control 1, or Control 2)
• Click the Restore Default Thresholds button to restore the vertical and horizontal
bars to their original position, which are indicated by the numbers next to the bars
•Select the C(t) Display Mode to view the data as threshold levels. Select RFU
Display Mode to view the data in relative fluorescence units at the selected cycle
•Select Normalize Data to normalize the RFU data shown in the chart and
spreadsheet
Normalization changes the data on the chart to a range from 0 to 1 on both axes. To normalize
the data, the plate must contain wells with “no template control” (NTC) sample types for both
Allele 1 and Allele 2. For this plot, the RFU data are normalized to the NTC values as a linear
combination of Allele 1- and Allele 2-specific RFUs. This plot is an effective way to present
RFU data.
The calculations for normalized RFU follows the formulas presented in Livak et al. (1995).
Where:
•A
1
represents RFU for Allele 1
•A
2
represents RFU for Allele 2
• represents the mean RFU
NTC
A1 + A2
represents the sum of RFUs for the NTC sample of Allele 1 and Allele 2
Allelic Discrimination tab Spreadsheet
The Allelic Discrimination spreadsheet at the top right side of the Allelic Discrimination tab
shows the information shown in Table 35.
QC Tab
Open the QC tab to quickly assess the quality of the experimental data based on the rules
defined in the QC tab in the User Preferences window (see “QC Tab” on page 128).
The software displays QC information in these layouts (Figure 95):
Table 35. Allelic Discrimination spreadsheet contents
Information Description
Well Well position in the plate
RFU1 or C(t)1 RFU or C(t) for Allele1
RFU2 or C(t)2 RFU or C(t) for Allele2
Call Identity of the allele, including automatic Allele 1, Allele 2,
Heterozygote, None, Unknown, Control 1, Control 2
Type Auto (Automatic) or Manual. Describes the way the call was
made. Automatic means the software selected the call.
Manual means the call was chosen by the user
Normalized A
1
A
1
A
1
A
2
xNTC
A1 A2+
()++
--------------------------------------------------------------
=

BIO RAD CFX96 Specifications

General

Temperature Uniformity	±0.4 °C
Software	CFX Maestro Software
Detection Method	Real-time PCR (qPCR)
Sample Capacity	96-well plate
Excitation Source	LED
Ramp Rate	Up to 5°C/sec
Heating Rate	5°C/sec
Sensitivity	Single copy detection
Power Requirements	100-240 V, 50/60 Hz
Temperature Range	0–100 °C