Qiagen PyroMark Q24 MDx, User Manual

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Operating Procedures
5-10 PyroMark Q24 MDx User Manual 01/2016
5.3.2 DNA amplification
Amplify the DNA to be analyzed by PCR using one of the
primers biotinylated. To receive valid analysis data, see
Appendix B.
5.3.3 Immobilizing the PCR product to beads
Biotinylated PCR products are immobilized on streptavidin-
coated Sepharose beads (Streptavidin Sepharose High
Performance, GE Healthcare).
1.
Gently shake the bottle with streptavidin-coated
Sepharose beads from side to side until a homogenous
solution is obtained.
2. Mix the total amount of streptavidin-coated Sepharose
beads (2 µl per sample) and Binding Buffer (40 µl per
sample) in a tube. Add high-purity water to a total
volume of 80 µl per well — including the PCR product to
be added in step 4.
The amount of water depends on the amount of PCR
product used. For example: If using 15 µl of PCR
product, 2 µl of beads, and 40 µl of Binding Buffer,
23 µl of high-purity water must be added.
3. Add the solution prepared in step 2 to a 24-well PCR
plate or strips.
4. Add 5–20 µl of a well-optimized, biotinylated PCR
product to each well of the PCR plate (or strips)
according to the plate setup (see Section 5.2.3).
Note : When using the PyroMark PCR Kit, 5–10 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 40 RLU in the Pyrogram.
Note : The total volume per well should be 80 µl.
5. Seal the PCR plate (or the strips) using strip caps. Ensure
that no leakage is possible between the wells.
6. Agitate the PCR plate (or strips) constantly for at least
5–10 min using a mixer (1400 rpm).