Operator`s manual

26
LightCycler® 480 Gene Scanning Software
Preparing a LightCycler® 480 System Gene Scanning Experiment
PCR Parameters
PCR Parameters5
Data obtained from High Resolution Melting is only as good as the amplification product
being analyzed. Nothing is more critical to High Resolution Melting data than having ro-
bust amplification of a single product. The High Resolution Melting Dye binds all double-
stranded DNA present in a reaction. Specific amplification product, undesired amplifica-
tion product(s), and primer-dimer products all bind the dye with equal affinity; thus all
contribute to the overall fluorescence and melting profile.
To optimize the amplification, determine the best thermal cycling parameters. If you use
the LightCycler® 480 High Resolution Master to establish your LightCycler® 480 Gene
Scanning assay, you can use the following parameters in your initial experiments:
Setup
Detection Format
High Resolution Melting Dye
Programs
Program Name Cycles Analysis Mode
Pre-Denaturation 1 None
Amplification 45
1)
Quantification
High Resolution Melting 1 Melting Curves
6)
Cooling 1 None
Temperature Targets
Target [°C] Acquisition Mode Hold (hh:mm:ss) Ramp Rate (°C/s)
(96-well / 384-well)
Acquisitions
(per °C)
Pre-Incubation
95 None 00:10:00 4.4 / 4.8 -
Amplification
95 None 00:00:10 4.4 / 4.8 -
primer dependent
2)
None 00:00:10 2.2 / 2.5 -
72 Single 00:00:10 -
00:00:20
3)
4.4 / 4.8
High Resolution Melting
95 None 00:01:00 4.4 / 4.8
40
4)
None 00:01:00 2.2 / 2.5
60
5)
None 00:00:01 1 -
95
5)
Continuous - - 25
Cooling
40 None 00:00:10 4.4 / 4.8 -