PyroMark Q24 Accessories and Reagents

For optimal sample preparation and Pyrosequencing analysis using the PyroMark Q24.

Features

  • Reliable quantification of genetic or epigenetic DNA modifications
  • Solutions for quick and easy sample handling for successful results
  • Dedicated products for each platform and each step in the workflow
  • High-quality optimized reagents enable reliable Pyrosequencing results

Products

PyroMark Q24 Accessories and Reagents are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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PyroMark OneStep RT-PCR Kit (200)

Cat. No. / ID: 978803

For 200 reactions: QIAGEN OneStep RT-PCR Enzyme Mix, optimized QIAGEN OneStep RT-PCR Buffer, 10x CoralLoad Concentrate, 5x Q-Solution, dNTP Mix, and RNase-Free Water
€ 687,00
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PyroMark OneStep RT-PCR Kit (50)

Cat. No. / ID: 978801

For 50 reactions: QIAGEN OneStep RT-PCR Enzyme Mix, optimized QIAGEN OneStep RT-PCR Buffer, 10x CoralLoad Concentrate, 5x Q-Solution, dNTP Mix, and RNase-Free Water
€ 232,00
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PyroMark Denaturation Sol. (500 ml)

Cat. No. / ID: 979007

For denaturation of double-stranded PCR product into single-stranded DNA template
€ 66,80
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PyroMark Q24 Validation Oligo

Cat. No. / ID: 979204

For performance confirmation of system
€ 716,00
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PyroMark PCR Kit (200)

Cat. No. / ID: 978703

For 200 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water.
€ 292,00
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PyroMark Q24 Cartridge (3)

Cat. No. / ID: 979202

Reusable dispensing tool for nucleotide and reagent delivery, for use with PyroMark Q24 and PyroMark Q24 Advanced
€ 517,00
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PyroMark Binding Buffer (200 ml)

Cat. No. / ID: 979006

For binding of biotinylated PCR product to Sepharose beads
€ 67,10
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PyroMark Q24 Plate (100)

Cat. No. / ID: 979201

24-well plate for samples, for use with PyroMark Q24 and PyroMark Q24 Advanced; 100 plates in each package
€ 337,00
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PyroMark PCR Kit (800)

Cat. No. / ID: 978705

For 800 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water
€ 971,00
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PyroMark Vacuum Prep Filter Probe (100)

Cat. No. / ID: 979010

Reusable filter probes for PyroMark Vacuum Workstation Q96 and Q24
€ 358,00
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PyroMark Control Oligo

Cat. No. / ID: 979203

For installation check of system
€ 344,00
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PyroMark Q24 Vacuum Prep Troughs (12)

Cat. No. / ID: 979206

Reusable plastic troughs for use with all PyroMark Q24 Vacuum Workstations
€ 39,40
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PyroMark Annealing Buffer

Cat. No. / ID: 979009

For annealing of sequencing primer to DNA template
€ 66,20
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PyroMark Gold Q24 Reagents (5 x 24)

Cat. No. / ID: 970802

Nucleotides, enzyme, and substrate solutions, intended for use with PyroMark Q24
€ 371,00
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PyroMark Wash Buffer (conc., 200 ml)

Cat. No. / ID: 979008

For use with the PyroMark Q24 Vacuum Workstation and PyroMark Q96 Vacuum Workstation to wash and neutralize the immobilized DNA
€ 66,80
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PyroMark Q24 Vacuum Workstation (220 V)

Cat. No. / ID: 9001518

Vacuum Workstation for preparing 24 samples in parallel from PCR product to single-stranded template

Product Details

The PyroMark Q24 uses proven Pyrosequencing technology for real-time, sequence-based detection and quantification for genetic analysis and epigenetic methylation studies. PyroMark Q24 Accessories and Reagents are high-quality reagents and products that simplify and streamline the Pyrosequencing workflow and enable successful results. PyroMark Q24 Accessories and Reagents are designed specifically for use with the PyroMark Q24 instrument.

Resources

Kit Handbooks (6)
For performing Pyrosequencing reactions on the PyroMark Q24
For use with PyroMark Q48 Autoprep, PyroMark Q24 Advanced, PyroMark Q24, PyroMark Q96 ID and PyroMark Q96 MD systems
Application Notes (2)
A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.
Analysis Software (5)
BRAF Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
RAS Extension Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
KRAS Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.6
For use with PyroMark Q24 Software version 2.0.8
GIST Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
EGFR Plug-in 1.3
SOFTWARE (2MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8

Publications

Genetic diagnostics of functional variants of the human dopamine D2 receptor gene.
Doehring A; Kirchhof A; Lötsch J;
Psychiatr Genet; 2009; 19 (5):259-68 2009 Oct PMID:19512960
[Quantitative pyrosequencing of heterozygous single nucleotide polymorphisms for rapid diagnosis of Down's syndrome].
Liu XQ; Wu HP; Bu Y; Zou BJ; Chen ZY; Zhou GH;
Zhonghua Yi Xue Yi Chuan Xue Za Zhi; 2009; 26 (3):331-5 2009 Jun PMID:19504451
Association between single nucleotide polymorphisms of surfactant protein D and allergic rhinitis in Chinese patients.
Deng YQ; Tao ZZ; Kong YG; Xiao BK; Chen SM; Xu Y; Wang Y; He Q;
Tissue Antigens; 2009; 73 (6):546-52 2009 Jun PMID:19493231
Global and gene-specific promoter methylation changes are related to anti-B[a]PDE-DNA adduct levels and influence micronuclei levels in polycyclic aromatic hydrocarbon-exposed individuals.
Pavanello S; Bollati V; Pesatori AC; Kapka L; Bolognesi C; Bertazzi PA; Baccarelli A;
Int J Cancer; 2009; 125 (7):1692-7 2009 Oct 1 PMID:19521983
BCL2-938C>A and CALCA-1786T>C polymorphisms in aseptic loosened total hip arthroplasty.
Wedemeyer C; Kauther MD; Hanenkamp S; Nückel H; Bau M; Siffert W; Bachmann HS;
Eur J Med Res; 2009; 14 (6):250-5 2009 Jun 18 PMID:19541585

FAQ

Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog no 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the catalog no 974203.

FAQ ID -2850
Can I order the nucleotides from PyroMark Gold Reagents separately?
The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.
FAQ ID -2827
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3-5 bases can be resolved depending on the sequence context and base. If it is possible sequencing of a homopolymer of more than 3-5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
Does the Pyromark Assay Design or application software give any cycling conditions for individual assay primers or a PCR setup pipetting scheme?
The PyroMark Assay Design and application software do not support PCR setup with a pipetting scheme or PCR cycling conditions. General recommendations how to setup and optimization of the PCR reaction are contained in the PyroMark PCR handbook.
FAQ ID -2862
What kind of shaker should be used for the pyrosequencing binding step?
Shaking conditions are 1400 rpm at room temperature. Optimal results are obtained with 2mm orbital diameter.
FAQ ID -2837
How many times can the cartridges for Pyromark Q24 or PyroMark Q96 ID instruments are reused?
When cleaning and storing the cartridges properly the Q24 cartridge can be used up to 30 times and Q96 ID cartridge up to 20 times. The cartridge product sheet and PyroMark Q24/ID user manual contains guidelines how to clean the cartridges correctly.
FAQ ID -2863
What should be the single peak height for the PyroMark Control Oligo on the different PyroMark instruments?


PyroMark Q24: The mean single peak height is 95 +/- 55 RLU
PyroMark Q48: The mean single peak height is 70 +/- 40 RLU
PyroMark Q96 ID: The mean single peak height is 35 +/- 10 RLU
Pyromark Q96 MD: The mean single peak height should be at least 350 RLU

FAQ ID -2852
What is the use of the PyroMark Q24 Validation Oligo?
The PyroMark Q24 Validation Oligo was developed to check the performance of the PyroMark Q24 system. It consists of two biotinylated oligonucleotides that only differ in one position (A or G) of the sequence. By mixing different proportions of the two oligonucleotides in replicates the linearity, bias and reproducibility of the system can be determined.
FAQ ID -2855
Which kits can be used in combination with the PyroMark CpG Assays and PyroMark Custom Assays?
QIAamp/DNeasy Kits can be used for DNA isolation, EpiTect Bisulfite Kits for DNA conversion, PyroMark PCR Kit for PCR amplification, EpiTect Control DNA Set for PCR controls, and PyroMark Gold Q24 Reagents or PyroMark Gold Q96 Reagents for the sequencing reaction.

Depending on the platform used, the following reagent kits are required for Pyrosequencing:

PyroMark Q96 ID and MD: PyroMark Gold Q96 Reagents

PyroMark Q24: PyroMark Gold Q24 Reagents

PyroMark Q24 Advanced: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

PyroMark Q48 Autoprep: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

FAQ ID -2822
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which serve as template. Perform accurate sequencing controls (e.g. PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
What is the concentration of PyroMark Control Oligo?
PyroMark Control Oligo has a concentration of 20µM and is delivered in a volume of 50µl. Two tubes of 10x dilution buffer (2x 1.7ml) are delivered with the control oligo.
FAQ ID -2846
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
Which heating block is recommended for the pyrosequencing annealing step?
A heating block that can heat up to 80-90 °C is recommended. A solid block is preferable. For the PyroMark Q24 the surface area must be 50mm x 60 mm and for Pyromark Q96 ID/MD 120mm x 80 mm.
FAQ ID -2838
What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.

 

For PyroMark Q24 and PyroMark Q96 MD the final concentration of the sequencing primer is 0.3µM and for PyroMark Q96 ID 0.4µM.


The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8µM, but may be adapted to optimize assays.

 

FAQ ID -2826
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
Can PyroMark Gold reagents be vortexed?
Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.
FAQ ID -2844
How accurate and reliable is PyroMark Q24 in mutation analysis?

PyroMark Q24 uses Pyrosequencing technology for mutation analysis and provides a built-in quality control in each run. By sequencing nucleotides flanking the mutation of interest, the researcher gets confirmation that the assay was made at the correct position. In addition, blank dispensations are included in the assay providing a negative control of the run. 

FAQ ID -2094
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878