Operator`s manual

24
LightCycler® 480 Gene Scanning Software
Preparing a LightCycler® 480 System Gene Scanning Experiment
PCR Primers
PCR Primers3
Design PCR primers that have annealing temperatures around 60°C and produce ►
short amplicons (100–250 bp). Use a software package like Primer3 (see http://frodo.
wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) or LightCycler® Probe Design Software
2.0 for designing the primers.
Use primers that have been purified by HPLC.
To avoid primer-dimer formation, use relatively low primer concentrations (less ►
than 300 nM) in the experimental reactions.
Depending on the amount of specific product observed in the initial experiment, ►
try repeating the experiment with a series of primer dilutions, increasing or de-
creasing the concentration in 0.1 µM steps. If initial production of the specific
product is robust, you might try lower concentrations of the primers. If initial
production of specific product is weak, try higher concentrations of the primers.
You do not need to use primers with GC clamps; they will not improve ►
High Resolution Melting.
BLAST the primer sequences to ensure they are specific for the target species and gene ►
(see http://www.ncbi.nlm.nih.gov/BLAST/).