Qiagen PyroMark Q96 Quick Start Manual

TechnicalInformation
PyroMark Q96 Vacuum Workstation quick-start guide
®
This Technical Information summarizes the immobilization and preparation of PCR products for Pyrosequencing
PyroMark Q96 Vacuum Workstation. Before beginning, carefully read Section 5.3.3 of the PyroMark Q96 ID User Manual
or Section 5.3.5 of the PyroMark Q96 MD User Manual and pay particular attention to the safety information.
Immobilizing the PCR products
1. Make a master mix according to the flowchart to the right.
Note: Before pipetting, gently shake the bottle of streptavidin-coated
Sepharose beads* to ensure a homogenous suspension.
®
2. Depending on sample volume and instrument type, pipet the correct
amount of master mix into each necessary well of a PCR plate to give a
total volume of 80 µl per well.
3. Add PCR product to each well, according to instrument type.
4. Seal the wells with strip caps and agitate the PCR plate at 1400 rpm for
5–10 min at room temperature (15–25°C) using an orbital shaker.
Separation of DNA strands and release of samples
into the PyroMark plate
1. Dilute sequencing primers with PyroMark Annealing Buffer (cat. no.
979009) and pipet into each necessary well of the instrument-specific
PyroMark plate. Position the plate on the workstation.
ID: 40 µl sequencing primer at 0.4 µM in the PyroMark Q96 Plate Low
MD: 12 µl sequencing primers at 0.3 µM in the PyroMark Q96 HS Plate
2. Fill the workstation troughs according to the diagram to the right.
3. Start the pump and apply vacuum to the tool by opening the switch.
4. Flush the filter probes with high-purity water (Milli-Q
or equivalent) in the "Parking" trough (P). Refill the trough with fresh
high-purity water for use in step 12.
5. Position the PCR plate on the workstation. Ensure that both plates are
in the same orientation as when the samples were loaded.
* Streptavidin Sepharose High Performance (34 μm, 5 ml, GE Healthcare). Check the lot
number of the Streptavidin Sepharose High Performance. For lot number 10057037
or higher, use 1.5 µl (ID) or 1 µl (MD) in the master mix. For lot numbers lower than
10057037, use 3 µl (ID) or 2 µl (MD). This is not a complete list of suppliers and does
not include many important vendors of biological supplies.
Beads*
ID:
1.5 or
3 µl/sample
MD:
1 or
2 µl/sample
ID: 5–20 µl of PCR product to each well
MD: 5–10 µl of PCR product to each well
Preparing the master mix to immobilize the PCR product.
90 ml
Denaturation
Solution
18.2 MΩ x cm
®
110 ml
70%
ethanol
Sample & Assay Technologies
®
Binding buffer High-purity water
ID: ID:
40 µl/sample 17 or 18.5–
33.5µl/sample
MD: MD:
40 µl/sample 28 or 29–
34 µl/sample
ID: 60 – 75 µl/well
MD: 70– 75 µl/well
(Total volume = 80 µl/well)
180 ml
high-purity
water
P
2 4
1 3
PCR plate PyroMark plate
using the
110 ml
high-purity
water
110 ml
Wash
Buffer