Immobilization Of The Pcr Product To Beads - Qiagen PyroMark Q96 ID User Manual

Operating Procedures
5.4.3

Immobilization of the PCR product to beads

Biotinylated PCR products are immobilized on streptavidin-
coated Sepharose beads (Streptavidin Sepharose High
Performance, GE Healthcare). For each sample the final
immobilization volume is 80 µl. The reaction setup is shown
in the table below.
Component
Biotinylated PCR
product
Streptavidin
Sepharose HP*
PyroMark Binding
Buffer
High-purity water †
Total volume
* Check the lot number of the Streptavidin Sepharose HP. For lot number 10057037 or higher use
1.5 µl. For lot numbers lower than 10057037, use 3 µl.
†
The volume of water depends on the amount of PCR product used.
Allow all reagents and solutions to equilibrate to room
temperature (15–25°C) before starting. All steps are
performed at room temperature, unless stated.
1. Gently shake the bottle with streptavidin-coated
2. Prepare a mixture of Streptavidin Sepharose beads,
3. Add the mixture prepared in step 2 to the wells of a 96-
4. Add 5–20 µl of a well-optimized, biotinylated PCR
5-8
Volume per sample
For lot number
10057037 or higher
5–20 µl
1.5 µl
40 µl
Variable †
80 µl
Sepharose beads from side to side until a homogenous
solution is obtained.
Binding Buffer, and high-purity water sufficient for all
samples to be analyzed.
well PCR plate according to the plate setup (see Section
5.3.1).
product to each well used.
Note: The total volume per well should be 80 µl.
PyroMark Q96 ID User Manual 01/2016
For lot numbers
lower than 10057037
5–20 µl
3 µl
40 µl
Variable †
80 µl