PyroMark Buffers and Solutions

For optimal preparation and analysis of template DNA by Pyrosequencing

Features

    undefined

    ✓ 24/7 automatic processing of online orders

    ✓ Knowledgeable and professional Product & Technical Support

    ✓ Fast and reliable (re)-ordering

    PyroMark Denaturation Sol. (500 ml)

    Cat. No. / ID: 979007

    For denaturation of double-stranded PCR product into single-stranded DNA template
    Buffer typeType
    Denaturation
    Binding
    Annealing
    Wash
    PyroMark Buffers and Solutions 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

    Generating Pyrosequencing templates from PCR amplicons is fast and straightforward with the PyroMark vacuum workstation. These buffers and solutions are specifically designed for this purpose. PyroMark Binding Buffer facilitates binding of biotinylated PCR amplicons to streptavidin-coated Sepharose beads. These beads are then immobilized with the vacuum tool of the workstation. PyroMark Denaturation Solution gently separates the complementary strand from the biotin-tagged strand destined to become the Pyrosequencing template, and PyroMark Wash Buffer ensures that only the biotin-tagged, single-stranded DNA remains immobilized to the vacuum tool. Finally, when the single-stranded DNA is released into reaction vessels containing the Pyrosequencing primer, hybridization of primer and template is enhanced by PyroMark Annealing Buffer.

    Resources

    Safety Data Sheets (2)
    Download Safety Data Sheets for QIAGEN product components.
    Download Safety Data Sheets for QIAGEN product components.

    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
    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
    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
    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
    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