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25
Preparing a LightCycler® 480 System Gene Scanning Experiment
PCR Reagents
PCR Reagents4
Hot-start PCR techniques are strongly recommended for High Resolution Melting
applications, since they avoid the formation of non-specific amplification products at the
beginning of the reaction.
Roche Applied Science provides a convenient, 2× concentrated master mix for such hot
start procedures. This LightCycler® 480 High Resolution Master contains FastStart Taq
DNA Polymerase and the High Resolution Melting Dye in a reaction buffer that contains
no MgCl
2
. The Master is compatible with additives (e.g., DMSO) that enhance amplifica-
tion of GC-rich sequences.
FastStart Taq DNA Polymerase is a chemically modified form of thermostable recombi-
nant Taq DNA polymerase that shows no activity at temperatures up to 75°C. The enzyme
is active only at high temperatures, where primers do not bind non-specifically. The en-
zyme is completely activated (by removal of blocking groups) during a single pre-incuba-
tion step (95°C, 5 minutes) before cycling begins.
A separate 25 mM MgCl
2
stock solution, supplied with the Master, allows you to easily
optimize the Mg
2+
concentration. Determining the optimal MgCl
2
concentration is
essential to ensure both the specificity and robustness of the PCR. The optimum MgCl
2
concentration for a LightCycler® 480 High Resolution Melting assay may vary from
1.5 to 3.5 mM. Therefore, we strongly recommend that you titrate the MgCl
2
concentra-
tion in the reaction between 1.5 and 3.5 mM (in 0.5 mM steps) when establishing a new
assay. Determine the specificity of each PCR by agarose gel electrophoresis.
The amount of MgCl
2
in a reaction will affect the shape and T
m
of the melting profile.
In some cases, fragments that melt with a single inflection at low MgCl
2
concentrations
may show multiple inflections at high MgCl
2
. While we observed this infrequently, you
should be aware of this possibility. As a general rule, at higher MgCl
2
concentrations,
a fragment will melt with a slightly higher overall T
m
.