Bio-Rad Precision Melt Analysis Software User Manual

AMPLIFICATION

Precision Melt Analysis™ Software
Quick Guide

Precision Melt Analysis software imports
and analyzes data files generated from a

™,

CFX96
Deep Well
CFX384 Touch
system to genotype samples based on the
thermal denaturation properties of
double-stranded DNA.

Installing Precision Melt
Analysis Software

A computer with either a Windows XP or
Windows 7 operating system is required
to run the software. The software must be
installed on the computer by a user with
administrative privileges.

To install the Precision Melt Analysis software:

1. Place the Precision Melt Analysis software

CFX96 To u ch™, CFX96 Touch

™

, CFX Connect™, CFX384™, or

™

real-time PCR detection

CD in the computer’s CD drive. The software
launch page should appear automatically.

Fig. 1. Welcome screen.

Fig. 2. Desktop software icon.

2. Click Next on the software launch page

(Figure 1).

3. Follow the instructions onscreen to
complete the installation.

4. If the launch page does not appear
automatically, click CD drive, then
open and follow the instructions in the
Readme.txt file.

5. After installation, launch the software
by double clicking the desktop icon
(Figure 2), or select Start > Programs >
Bio-Rad > Precision Melt Analysis.

Melt Calibration

Before Precision Melt Analysis software can analyze data generated on
a CFX96, CFX96 Touch, CFX96 Touch Deep Well, CFX Connect, CFX384,
or CFX384 Touch real-time PCR detection system, a melt calibration must
be performed. The procedure requires running an experiment and then
importing the data into Precision Melt Analysis software to generate a melt
calibration file. The software is shipped with a calibration kit that includes
the following materials:

n 

Melt calibration DNA standard

n

Melt calibration primers

n

Precision melt supermix (catalog #172-5110)

Additional Materials Required

In addition to the components provided in the melt calibration kit,
the following materials are required:

n

PCR-grade tubes

n

Nuclease-free water

n 

Microseal® 'B' adhesive seals, optically clear (catalog #MSB-1001)

n 

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

n

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, CFX96 Touch Deep Well, or
CFX Connect system

Preparing a Melt Ca libration Plate

1. Add the required volume of each component to an appropriately
sized tube (Table 1).

2. Cap the tube and gently mix the reaction components by vortexing.

3. Briefly centrifuge the tube to remove air bubbles and collect
contents at the bottom of the tube.

4. Add the appropriate volume of the mixture into each well of a
reaction plate:

n 

For a CFX96, CFX96 Touch, CFX96 Touch Deep Well, or
CFX Connect system, add 20 µl of the reaction mixture to each
well of a 96-well plate

n

For a CFX384 or CFX384 Touch system, add 10 µl of the reaction
mixture to each well of a 384-well plate

5. Seal the reaction plate with a Microseal 'B' adhesive seal. Centrifuge
the plate at 1000 x g for 2 min to move all the reaction components
to the bottom of the wells.

Performing a Melt Calibration Experiment

A melt calibration experiment is run on a CFX96, CFX96 Touch,
CFX96 Touch Deep Well, CFX Connect, CFX384, or CFX384 Touch real­time PCR detection system and analyzed using CFX Manager

™

software.

To run a melt calibration to generate a melt calibration data file:

Table 1. Reac tion setup for a melt calibration plate.

Volume for Volume for
Component CFX96 System CFX384 System

Precision melt 1,200 µl 2,250 µl
supermix

Melt calibration 120 µl 450 µl
DNA standard

Melt calibration 14.4 µl 27 µl
primers

Nuclease-free water 1,065.6 µl 1,773 µl

Total 2,400 µl 4,500 µl

Fig. 3. Startup Wizard in CF X Manager software.

Table 2. Melt calibration PCR protocol.

Number
Cycling Step Temperature T ime of Cycles

Enzyme activation 98°C 2 min 1

Denaturation 98°C 5 sec

Annealing/extension 55°C 10 sec

95°C 1 min 1

70°C 1 min 1

Melt curve 70– 95°C 10 sec/step 1

(in 0.2°C
increments)

35

1. Turn on the CFX 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
(Figure 3) to launch the Run Setup window.