Bio-Rad Precision Melt Analysis™ Software - Manual

Melt Calibration Kit
Catalog #184-5020
Kit Contents
The following materials are provided in the melt calibration kit:
 Melt calibration DNA standard
 Melt calibration primers
 Precision melt supermix (catalog #172-5110)
Storage and Stability
Precision melt supermix is stable until the date indicated when stored in a constant temperature freezer at –20°C. For convenience, it
may be refrozen for longer-term storage.
Product Overview
Before Precision Melt Analysis™ software can analyze data generated on a CFX96™, CFX96 Touch™, CFX384™, CFX384 Touch™,
or CFX Connect™ real-time PCR detection system, a melt calibration must be performed. The calibration is required regardless of the
intercalating dye that will be used in the experiments, including SYBR
® Green I.
Additional Materials Required
In addition to the components provided in the melt calibration kit, the following materials are required:
 PCR-grade tubes
 Nuclease-free water
 Microseal ® ‘B’ adhesive seals, optically clear (catalog #MSB-1001)
 Hard-Shell ® thin-wall 384-well skirted PCR plates with clear shell and white wells (catalog #HSP-3805) for use with a CFX384,
or CFX384 Touch system
 Hard-Shell ® thin-wall 96-well skirted PCR plates with white shell and clear wells (catalog # HSP-9601) or white wells (catalog
#HSP-9655) for use with a CFX96, CFX96 Touch, or CFX Connect system
Preparing the Melt Calibration Plate
1. Add the required volume of each component to an appropriately sized tube (Table 1).
Table 1. Reaction setup for a melt calibration plate.
Component
Volume for CFX96,
CFX96 Touch, or CFX
Connect System
Volume for CFX384 or
CFX384 Touch System
Precision melt supermix 1,200
l 2,250 l
Melt calibration DNA standard 120 l 450 l
Melt calibration primers 14.4
l 27 l
Nuclease-free water 1,065.6
l 1,773 l
Totals 2,400 l 4,500 l
2. Cap the tube and mix the reaction components gently by vortexing.
3. Briefly centrifuge the tube to remove air bubbles and collect contents at the bottom.
4. Add the appropriate volume of the mixture into each well of a reaction plate.
 For a CFX96, CFX96 Touch, or CFX Connect system, add 20 μl of the reaction mix to each well of the 96-well plate
 For a CFX384 or CFX384 Touch system, add 10 μl of the reaction mix to each well of the 384-well plate
5. Seal the reaction plate with Microseal ‘B’ adhesive film. Centrifuge the plate to move all the reaction components to the
bottom of the wells.
Performing the Melt Calibration Experiment
The melt calibration experiment is run on a CFX96, CFX96 Touch, CFX384, CFX384 Touch, or CFX Connect system and analyzed
using CFX Manager™ software.
To run the melt calibration and generate a melt calibration data file:
1. Turn on the CFX96, CFX384, or CFX Connect system.
2. Double-click the CFX Manager software desktop icon to launch the software.
3. Select Create a New Experiment from the list of options in the Startup Wizard. Click OK to launch the Experiment Setup
window.
4. In the Protocol tab, select Create New to open the Protocol Editor.
5. Create the following protocol (Table 2).