Qiagen PyroMark Q96 ID User Manual page 99

These include the QIAprep
purification, the QIAamp
purification of genomic DNA and viral nucleic acids, and the
RNeasy
sources. For more information about QIAprep, QIAamp,
DNeasy, and RNeasy products, contact one of our Technical
Service Departments (see back cover) or visit
www.qiagen.com.
Quality of starting template when performing CpG
assays
Critical parameters for a successful PCR using bisulfite-
treated DNA templates include complete bisulfite conversion
and DNA fragments that are long enough for PCR. EpiTect
Plus Bisulfite Kit provides a fast and reliable procedure for
efficient bisulfite conversion and a unique DNA Protect Buffer
prevents DNA fragmentation during the bisulfite conversion
reaction. For more information about EpiTect products,
contact one of our Technical Service Departments (see back
cover or visit www.qiagen.com).
Quantity of starting template
The annealing efficiency of a primer to the template is an
important factor in PCR. Owing to the thermodynamic nature
of the reaction, the primer:template ratio strongly influences
the specificity and efficiency of PCR and should be optimized
empirically. If too little template is used, primers may not be
able to find their complementary sequences. Too much
template may lead to an increase in mispriming events.
PCR optimization
The PyroMark PCR Kit will produce satisfactory results in most
cases. However, if a higher Mg
we recommend using the 25 mM MgCl
PyroMark Q96 ID User Manual 01/2016
®
®
system for RNA preparation from a variety of
® system for rapid plasmid
and DNeasy ® systems for rapid
2+ concentration is required,
provided in the kit.
2
Appendix B
®
B-3