Qiagen PyroMark Q96 ID User Manual page 103

Pay attention to tips and warnings indicated by the red
icon and make suitable modifications when the icon
appears.
Validation of a new assay
Controls
All new assays have to be validated by the user. Use a
reference DNA sample when testing a new assay and ensure
that appropriate analysis parameters in the PyroMark Q96
Software are used. Interactions between primers or loops
formed on single-stranded DNA can serve as priming sites
for base incorporation by DNA polymerase. The following
controls should be included when an assay is analyzed for
the first time:
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Pyrograms from these controls should not show a peak after
any nucleotide addition.
PyroMark Q96 ID User Manual 01/2016
PCR without template DNA. This will show if the primers
interact to give a background signal in Pyrosequencing
reactions.
PCR with template DNA but with no sequencing primer.
This will show if the template can loop back on itself and
give a background signal in Pyrosequencing reactions.
Sequencing primer without any PCR product. This will
show if the sequencing primer can form duplexes or
hairpins and give background signal in Pyrosequencing
reactions.
Biotinylated primer without any PCR product. This will
show if the biotinylated primer can form duplexes or
hairpins and give background signal in Pyrosequencing
reactions.
Sequencing primer and biotinylated primer together
without PCR product. This will show if the sequencing
primer and the biotinylated primer can form duplexes
and give background signal in Pyrosequencing reactions.
Appendix B
B-7