Operator`s manual

27
Preparing a LightCycler® 480 System Gene Scanning Experiment
PCR Parameters
In case you do not know the melting temperatures of your PCR primers exactly, it is
recommended to apply a touchdown PCR protocol covering a range of annealing tem-
perature from 65 to 53°C. Modify the Temperature Targets of the Amplification program
as shown in the table below:
Target
(°C)
Acquisition
Mode
Hold
(hh:mm:ss)
Ramp Rate
(°C/s)
(96-well /
384-well)
Acquisi-
tions
(per °C)
Sec Target
(°C)
Step
Size (°C)
Step
Delay
(cycles)
95 None 00:00:10 4.4 / 4.8 - 0 0 0
65 None 00:00:10 2.2 / 2.5 - 53 0.5 1
72
3)
Single 00:00:10 -
00:00:20
4.4 / 4.8 - 0 0 0
1)
Number of cycles
45 cycles are suitable for most assays. If the assay is optimized and has steep amplifica-
tion curves and early crossing points (even when target concentrations are low), 40 cycles
should be sufficient. Reducing the number of cycles will reduce the time required for the
assay.
2)
Annealing temperature
Annealing temperature is the parameter that most influences specificity and robustness of
amplification. For initial experiments set the target temperature (i.e., the primer anneal-
ing temperature) 2°C below the calculated primer T
m
. The amount of specific product, the
presence/absence of undesirable side product, and the presence/absence of dimer product
in these experiments will dictate the best way to optimize this parameter. If the reaction
produces undesirable product, increase the annealing temperature. If amplification is not
robust, decrease the annealing temperature and/or increase the duration of the annealing
step.
3)
Elongation time
Calculate the exact elongation time required for your specific target by dividing the ampli-
con length by 25 (e.g., a 500 bp amplicon requires 20 s elongation time).
4)
Melting pre-hold step
This pre-hold temperature ensures that all PCR products have re-associated and encour-
ages heteroduplex formation.
5)
Melting interval
Actual melting conditions depend upon the amplicon. For initial experiments set a wide
melting interval, e.g., from 60 to 95°C. Once you have determined where the product will
melt, reduce the melting interval to approximately 25°C. Ensure that the melt program
starts at least 10°C before and ends at least 10°C after the expected T
m
value.
6)
Analysis mode
No special analysis mode for Gene Scanning assays is available. Gene Scanning experi-
ments are performed in standard Melting Curves analysis mode.