Bio-Rad Precision Melt Analysis™ Software - Manual (Page 17)

Precision Melt Analysis™ Software
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5. Seal the reaction plate with Microseal ‘B’ adhesive film. Centrifuge the melt calibration
plate to move all the reaction components to the bottom of the well.
Performing the Melt Calibration Experiment
The melt calibration experiment is run on a CFX96, CFX Connect, or CFX384 real-time PCR
system using the prepared melt calibration plate.
To run the melt calibration to generate a melt calibration data file
1. Turn on the real-time PCR system.
2. Double-click the CFX Manager software desktop icon to launch the software.
3. Select User-defined from the Run setup tab of the Startup Wizard to launch the Run
Setup window.
4. In the Protocol tab, select Create New to open the Protocol Editor.
5. Create the following protocol (Table 6).
6. Click OK to save the protocol and return to the Experiment Setup window.
Or, click the Protocol tab
Click Select Existing > Sample Files > Melt Calibration Folder, select the appropriate protocol
name based on your instrument and click Open
• For a 96-well system, select Melt Calibration Protocol_96
• For a 384-well system, select Melt Calibration Protocol_384
7. Click the Plate tab.
8. Click Select Existing > Sample Files > Melt Calibration folder, select the plate name
based on the instrument and click Open.
• For a 96-well system, select Melt Calibration Plate_96 wells_Clear, or Melt
Calibration Plate_96 wells_White
• For a 384-well 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 checkbox to
the left of the instrument name.
11.Click Open Lid, then load the melt calibration plate into the instrument.
12.Click Close Lid.
Table 6. Melt calibration protocol
Cycling Step Temperature Time # of Cycles
Enzyme activation 98°C 2 min 1
Denaturation 98°C 5 sec 35
Annealing/Extension 55°C 10 sec
95°C 1 min 1
55°C 1 min 1
Melt curve 70–95°C (in 0.2°C
inc.) 10 sec/step 1