Operator`s manual

23
Preparing a LightCycler® 480 System Gene Scanning Experiment
Sample Material
Sample Material2
Because a LightCycler® 480 System Gene Scanning experiment involves comparing melt-
ing profiles from independent PCR reactions, it is crucial to minimize reaction-to-reac-
tion variability. Standardizing the template DNA is one means of minimizing variability.
Follow these guidelines when preparing or handling template DNA:
Use isolation and storage procedures that minimize the potential for sample degrada- ►
tion. Avoid procedures that can introduce excessive amounts of inhibitors (e.g., due to
ethanol carry-over).
If extraction is required, use the same extraction procedure to prepare all samples to ►
be analyzed via High Resolution Melting. This eliminates any subtle differences that
might be introduced by the reagent components in the final elution buffers of different
extraction procedures.
For reproducible isolation of nucleic acids use:
Either the MagNA Pure LC Instrument or the MagNA Pure Compact Instrument ►
together with a dedicated nucleic acid isolation kit (for automated isolation) or
A high pure nucleic acid isolation kit (for manual isolation), e.g., the High Pure ►
PCR Template Preparation Kit.
For details see the Roche Applied Science Biochemicals catalog or home page,
http://www.roche-applied-science.com.
Resuspend all DNA samples in the same buffer, quantify them using spectrophotome- ►
try, and adjust them to the same concentration with the resuspension buffer. Salts
affect DNA melting behavior, so it is important that the concentrations of buffer, Mg
2+
and other salts in the reaction mix are as uniform as possible for all samples.
Use the same amount of template in each reaction. The recommended amount is ►
5 to 30 nanograms of template DNA in a 20 µl reaction volume, which should produce
amplification plots with a Cp value of no more than 30 cycles. Products that reach this
threshold at higher Cps (due to insufficient amounts of starting template or template
degradation) typically produce variable High Resolution Melting results due to am-
plification artifacts.
If you are using archival genomic DNA, repurify the DNA by binding it to silica ( ► e.g.,
with the High Pure PCR Template Preparation Kit from Roche Applied Science) and
eluting it into a fresh buffer before using it in a Gene Scanning experiment. This will
eliminate artifacts caused by sublimation (the direct transition of frozen material to
gas), which frequently occurs in such samples and concentrates salts and other mate-
rial that affect both amplification and High Resolution Melting.