Qiagen PyroMark Q96 User manual

TechnicalInformation
Sample & Assay Technologies
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®18.2 MΩ x cm
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.
PyroMark®Q96 Vacuum Workstation quick-start guide
This Technical Information summarizes the immobilization and preparation of PCR products for Pyrosequencing®using the
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.
Preparing the master mix to immobilize the PCR product.
Beads*
ID:
1.5 or
3µl/sample
MD:
1or
2 µl/sample
Binding buffer
ID:
40 µl/sample
MD:
40 µl/sample
ID: 5–20 µl of PCR product to each well
MD: 5–10 µl of PCR product to each well
(Total volume = 80 µl/well)
High-purity water
ID:
17 or 18.5–
33.5µl/sample
MD:
28 or 29–
34 µl/sample
ID: 60–75µl/well
MD: 70–75µl/well
180 ml
high-purity
water
4
P
2
31
110 ml
high-purity
water
90 ml
Denaturation
Solution
110 ml
70%
ethanol
PCR plate PyroMark plate
110 ml
Wash
Buffer
* 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.

Sample & Assay Technologies
Trademarks: QIAGEN®, PyroMark®, Pyrosequencing®(QIAGEN Group); Sepharose®(GE Healthcare); Milli-Q®(Millipore Corporation).
1074529 12/2012 © 2012 QIAGEN, all rights reserved.
6. With the vacuum switch ON, lower the vacuum tool into the wells of the PCR plate for 15 s to
capture the beads with PCR product.
7. With vacuum ON, flush the tool with 70% ethanol (trough 1) for 5 s.
8. With vacuum ON, flush the tool with Denaturation Solution (trough 2) for 5 s.
8. With vacuum ON, flush the tool with Wash Buffer (trough 3) for 10 s.
10. With vacuum ON, raise the tool to beyond 90° vertical for 5 s.
11. Align the vacuum tool with the PyroMark plate and switch the vacuum OFF. Lower the vacuum
tool into the wells and gently shake from side to side to release the beads.
12. With the vacuum OFF, agitate the vacuum tool in high-purity water (trough 4) for 10 s.
13. With vacuum ON, flush the filter probes with high-purity water (trough P) for 5 s.
14. Raise the vacuum tool to beyond 90° vertical for 5 s, then switch the vacuum OFF and store
the tool in the “Parking” position.
CAUTION:
PyroMark Denaturation
Solution contains sodium
hydroxide, which is an
eye and skin irritant.
Always wear a suitable
lab coat, disposable
gloves, and protective
goggles. For more
information, see the
PyroMark Q96 ID User
Manual or the PyroMark
Q96 MD User Manual
(and the MSDS).
Annealing sequencing primers to DNA strands
Place the PyroMark Q96 Plate Low in a prewarmed PyroMark Q96 Sample Prep Thermoplate Low.
Alternatively, place the PyroMark Q96 HS Plate in a prewarmed PyroMark Q96 HS Sample Prep
Thermoplate. Heat the Pyrosequencing samples on a heating block at 80°C for 2 minutes. Remove
the plate from the thermoplate and allow the samples to cool to room temperature (15–25°C) for
at least 5 minutes. The cooled plate can now be processed.
Cleaning the vacuum workstation
Liquid waste and solutions remaining in the troughs of the vacuum workstation should be
appropriately discarded at the end of the day. For details, see your instrument user manual.
For up-to-date licensing information and product-specific disclaimers, see the PyroMark Q96 ID User Manual or PyroMark
Q96 MD User Manual. The PyroMark Q96 ID User Manual and PyroMark Q96 MD User Manual are available at
www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
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