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PyroMark Q96 ID Accessories and Reagents

For optimal sample preparation and Pyrosequencing analysis using PyroMark Q96 ID

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
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PyroMark Gold Q96 SQA Reagents (1 x 96)

Cat. No. / ID: 972812

For performing Pyrosequencing reactions on the PyroMark Q96 ID
ReagentAccessoriesSoftwareKitAssayInstrument
Q96 Reagents
Consumables
Q96 Sample Prep Thermoplate Low
PyroMark Software
PyroMark Kits
Assays and Tests
Buffers
Vacuum Workstation
Type
Gold Q96 SQA Reagents
Gold Q96 Reagents
Gold Q96 CDT Reagents
PyroMark Q96 ID Accessories and Reagents are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

The PyroMark Q96 ID uses proven Pyrosequencing technology for real-time, sequence-based detection and quantification for genetic analysis and epigenetic methylation studies. PyroMark Q96 ID Accessories and Reagents are high-quality reagents and products that simplify and streamline the Pyrosequencing workflow and enable successful results. PyroMark Q96 ID Accessories and Reagents are designed specifically for use with PyroMark Q96 ID instruments.

Applications

PyroMark Q96 ID Accessories and Reagents enable Pyrosequencing analysis with the PyroMark Q96 ID instrument for applications such as:
  • HRM
  • Methylation analysis
  • Mutation analysis
  • SNP analysis

Resources

Safety Data Sheets (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.
Application Notes (2)
A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.
Kit Handbooks (5)
For use with PyroMark Q48 Autoprep, PyroMark Q24 Advanced, PyroMark Q24, PyroMark Q96 ID and PyroMark Q96 MD systems
For performing Pyrosequencing reactions on the PyroMark Q96 ID, PyroMark Q96 MD, and PyroMark Q96 MD Automated

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
Pyrosequencing enhancement for better detection limit and sequencing homopolymers.
Gong M; Foo SH; Lin L; Liu ET; Gharizadeh B; Goel S;
Biochem Biophys Res Commun; 2010; 401 (1):117-23 2010 Sep 15 PMID:20833128
Rapid molecular identification of pathogenic yeasts by pyrosequencing analysis of 35 nucleotides of internal transcribed spacer 2.
Borman AM; Linton CJ; Oliver D; Palmer MD; Szekely A; Johnson EM;
J Clin Microbiol; 2010; 48 (10):3648-53 2010 Aug 11 PMID:20702674
[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

FAQ

What is the meaning of the abbreviations NDT and CDT and what are the differences?

The PyroMark Q96 MD uses the NDT and CDT to dispense nucleotides, while the Q24 and Q96 ID use cartridges.

NDT means nucleotide dispensing tip. This is the original dispensing tip which is more sensitive to dust but dispenses lower nucleotide volumes and enables longer read length.

CDT means caplillary dispensing tip and is comparable to Q24 and Q96 ID cartridges. They dispense a higher volume but are easier in handling.

The NDT and CDT use two different tip holders which cannot be interchanged

 

 

 

FAQ ID -2841
In which format can PyroMark CpG Assays be ordered?
Pyromark CpG Assay can be ordered in tubes or on 96 well plates. PyroMark CpG Assays 96 well require a minimum order of 24 assays per plate.
FAQ ID -2824
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
Which end of the PCR primer for pyrosequencing should be biotinylated?
In Pyrosequencing, the 5' end should be biotinylated, regardless of whether the forward or reverse primer is biotinylated. You can order pyrosequencing primers here.
FAQ ID -2839
How many times can the CDTs, NDTs, and RDTs be used?
The PyroMark Q96 MD HS Capillary Tip and HS Reagent Tip  can be used up to 20 times and the PyroMark Q96 HS Nucleotide Tip up to 10 times.
FAQ ID -2865
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
What is included in a PyroMark Custom Assay?
The Pyromark Custom Assay includes a 10x PCR Primer Set (mixture of forward and reverse PCR Primer) and a 10x Sequencing Primer. Reagents for performing PCR and Pyrosequencing reaction are not included.
FAQ ID -2815
What are the features of PyroMark CpG Assays e.g. in terms of design, validation?
PyroMark CpG Assays are genome-wide, pre-designed methylation assays for pyrosequencing analysis. An optimized design algorithm was used for highly specific assay design and advanced CpG methalytion results.
FAQ ID -2821
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
Which PyroMark Gold Q96 Reagent should be used for which instrument and application?

PyroMark Gold Q96 Reagents:

  • PyroMark Gold Q96 SQA Reagents (1 x 96) should be used for performing SQA analyses on the PyroMark Q96 ID. It can also be used to supplement the PyroMark Gold Q96 Reagents (5 x 96) when long runs lead to a shortage of nucleotides.
  • PyroMark Gold Q96 Reagents (5 x 96) should be used for SNP and CpG analyses on the PyroMark Q96 ID and MD instruments. On the PyroMark Q96 MD, the kit contains enough for 15x96 plates.
  • PyroMark Gold Q96 CDT Reagents (6 x 96) should be used only with CDTs on the PyroMark Q96 MD instrument, for performing SNP and CpG anslyses. The nucleotides are pre-diluted for use with CDTs, so this is not to be used on ID instrument or with NDTs on the MD.
  • PyroMark Gold Q96 Reagents (50 x 96) should be used only with the PyroMark Q96 MDA (automated option) for SNP anslyses. Not for use with the PyroMark Q96 ID or non-automated MD.

See the PyroMark Gold Q96 Reagents Handbook for additional information.

FAQ ID -2836
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 are the PyroMark CpG Assays shipped and stored?
Pyromark CpG Assays are shipped lyophilized at ambient temperatures (20-25°C) and should be stored at -20°C either reconstituted or lyophilized. Repeated freeze-thaw cycles should be avoided. When stored under these conditions, the reconstituted product can be kept for at least 18 month from the date of receipt without reduction in performance.
FAQ ID -2816
How are the PyroMark CpG Assays reconstituted?
The PyroMark CpG Assay is reconstituted as a 10x PCR Primer Set in 550µl TE, pH 8.0 and the 10x Sequencing Primer is reconstituted in 1175µl Annealing Buffer if using the PyroMark Q24, 880µl if using the PyroMark Q96 ID, and 1175µl if using the PyroMark Q96 MD.
FAQ ID -2817
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
Does QIAGEN offer a design of Custom PyroMark CpG Assays?
No, QIAGEN does not design any Custom PyroMark CpG Assay. Customers have the possibility to order pre-designed, genome-wide PyroMark CpG Assays or order a user-designed assay (e.g. with the PyroMark Assay Design Software or assays known from previous projects or from the literature).
FAQ ID -2818
Will the primer sequence for the PyroMark CpG Assay be provided?
Primer sequences for PyroMark CpG Assays are not provided, they are proprietary.
FAQ ID -2823
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
Will dUTP in a PCR reaction affect pyrosequencing?
In general, dUTP/UNG treatment should work for pyrosequencing in order to reduce contamination risk with PCR amplicons from previous PCR’s.
FAQ ID -2843
Is the CpG software included in the PyroMark instruments to study methylation status?
The PyroMark Q24 software and the new PyroMark Q96 ID software version 2.5 support CpG analysis in the CpG mode. The ORACLE-based PyroMark Q96 ID software version and PyroMark Q96 MD software do not support CpG analysis. In this case an independent, additional software is needed which is the PyroMark CpG software version 1.0.
FAQ ID -2842
What is the purpose of the unmethylated and unconverted control DNA of the EpiTect PCR Control DNA Set?

The unmethylated and unconverted human control DNA of the EpiTect PCR Control DNA Set allows to check that primers designed for the specific detection of unmethylated and converted DNA (U-converted DNA), and for methylated, converted DNA (M-converted DNA) do not bind to untreated genomic DNA.*

In case bisulfite conversion was not complete, leaving certain unmethylated C residues unconverted, false positives would result if the primer specific for M-converted DNA binds to untreated gDNA.

This control DNA can also be used to check conversion efficiency during bisulfite treatment.

 

*(Summary of principle: Methylation of DNA occurs on cytosine residues, especially on CpG dinucleotides enriched in small regions of DNA. Incubation of target DNA with sodium bisulfite, using e.g. EpiTect Bisulfite Kits, results in conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines unchanged).

 

FAQ ID -2007
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
What is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200bp). This is critical especially for DNA from FFPE tissue which is often degraded by the fixation so that short fragments are easier to amplify. Moreover, the DNA suffers from harsh bisulfite treatment and might receive further double strand breaks. Therefore the amplicon size should be kept as short as possible.
FAQ ID -2825
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 do I set up a PyroMark CpG Assay?
All relevant information regarding PyroMark CpG Assay setup can be found on the Gene Globe website. The Sequence to Analyse and dispensation order should not be copied manually to create a new assay. Instead, the assay file should be downloaded from the web and opened in the PyroMark CpG software, PyroMark Q96  ID v2.5 (or higher) software, and PyroMark Q24 Software in order to keep important software settings.
FAQ ID -2814
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