Bio-Rad Precision Melt Analysis™ Software User Manual
Page 2
Table 2. Melt calibration PCR protocol.
Cycling Step
Temperature
Time
# of Cycles
Enzyme activation
98°C 2
min
1
Denaturation
98°C 5
sec
Annealing/extension
55°C 10
sec
35
95
C
1 min
1
70
C
1 min
1
Melt curve
70–95°C (in 0.2°C
increments)
10 sec/step
1
6. Click OK to save the protocol and return to the Experiment Setup window.
7. Click the Plate tab.
8. Click Select Existing > Sample Files > Melt Calibration Folder, select the appropriate plate name based on your instrument
and plate type, and click Open.
For a CFX96 or CFX Connect system, select Melt Calibration Plate_96 wells_Clear or Melt Calibration Plate_96
wells_White
For a CFX384 system, select Melt Calibration Plate _384 wells_White
9. Click the Start Run tab.
10. Select the instrument in the Start Run on Selected Blocks list by clicking the check box to the left of the instrument name.
11. Load the melt calibration plate into the instrument.
12. Click Start Run to begin running the experiment on the selected block.
13. At the prompt, save the name of the melt calibration data file as Melt Calibration_today’s date.
14. When the melt calibration run is complete, the data file is automatically processed and opened by CFX Manager software.
Check the data file to ensure all wells display a tight amplification and a single melt peak (Figure 1).
Fig. 1. Melt calibration data. A, amplification plot; B, melt curve plot with a single peak.
B
A
Importing a Melt Calibration File
The Precision Melt Analysis software is used to open data files that have been generated from an experiment performed on a CFX96,
CFX96 Touch, CFX384, CFX384 Touch, or CFX Connect real-time PCR detection system and analyzed using CFX Manager software.
To open and generate a melt calibration file using the Precision Melt Analysis software:
1. Launch the Precision Melt Analysis software by double-clicking on the Precision Melt Analysis software icon on the desktop.
2.
Click Tools > Import Melt Calibration from the Menu bar.
3. Choose the name of the melt calibration experiment data file (.pcrd extension) and click Open.
4. A window will appear indicating the calibration was successful.
5. Click OK to proceed and use Precision Melt Analysis software.
NOTICE TO PURCHASER: LIMITED LICENSE
Use of this product is covered by one or more of the following U.S. patents and corresponding patent claims outside the U.S.: 5,079,352, 5,789,224, 5,618,711, 6,127,155,
5,677,152 (claims 1 to 23 only), 5,733,258 (claims 1 and 6 only), 5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, and claims outside the U.S. corresponding to U.S.
Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of
product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus or system claims in U.S. Patent No. 6,814,934) and no right to
perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed
expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further
information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404,
U.S.A.
Hard-Shell PCR plates are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Nos. 7,347,977,
6,340,589, 6,528,302.
Bio-Rad Laboratories, Inc. is licensed by Biotium Inc. to sell reagents containing EvaGreen dye for use in real-time PCR, for research purposes only.
SYBR
®
is a registered trademark of Molecular Probes, Inc. EvaGreen is a registered trademark of Biotium, Inc.
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