Qiagen PyroMark Q96 ID User Manual page 101

Sample preparation
Use 5–20 µl of a 25 µl PCR for immobilization of PCR
amplicons to streptavidin-coated sepharose beads
(Streptavidin Sepharose High Performance, GE Healthcare)
according to the instructions in Section 5.4.3. This will be
equivalent to approximately 1–4 picomoles of PCR product,
depending on the PCR efficiency.
Note: When using the PyroMark PCR Kit, 20 µl of the PCR
product gives satisfactory Pyrosequencing results in most
cases. This volume should be adjusted to achieve single peak
heights of at least 20 RLU in the Pyrogram.
Pyrosequencing analysis
Use default settings in the software for all assay setups if not
otherwise stated.
Assay setup
AQ and CpG assays
When creating an AQ or CpG assay, the sequence to
analyze should contain a sufficient number of bases to
generate at least five nonvariable reference peaks. For
InDels, a few reference peaks should be included before the
variable position.
If the sequencing primer is placed immediately adjacent to
the position to analyze, include part of the sequence
following the variable position in the "Sequence to Analyze"
text box. Ensure that the last base in the text box is not part of
the variable position.
Blank dispensations are automatically generated by the
software and serve as built-in quality controls for the assay.
Never exclude the blank dispensations generated by the
software as they function as excellent indicators of unspecific
nucleotide incorporation. When manually generating a
dispensation order, include an appropriate number of blank
dispensations. If possible, start the dispensation order with
PyroMark Q96 ID User Manual 01/2016
Appendix B
B-5