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    EZ-96 DNA Methylation-Direct Kit


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    Cat # Name Size
    D5023 EZ-96 DNA Methylation-Direct Kit (Deep-well) 2 x 96 Rxns.
    D5022 EZ-96 DNA Methylation-Direct Kit (Shallow- well) 2 x 96 Rxns.

    Highlights

    • High-throughput 96-well bisulfite conversion of DNA directly from blood, soft tissue, cells, FFPE samples, and LCM samples.
    • Compatible with small sample inputs as few as 10 cells or 50 pg DNA.
    • DNA recovered by this bisulfite conversion kit is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.
    Description & Documents Specifications FAQ Components Citations

    Documents

    Supplemental Info

    Product Description

    The EZ-96 DNA Methylation-Direct Kit is a 96-well bisulfite conversion kit that features reliable and complete DNA bisulfite conversion directly from blood, tissue, and cells without the prerequisite for DNA purification. The increased sensitivity of this 96-well bisulfite conversion kit makes it possible to amplify bisulfite-converted DNA from as few as 10 cells or 50 pg DNA. Recovered bisulfite-converted DNA is ideal for PCR amplification for downstream analyses including restriction endonuclease digestion, sequencing, microarrays, etc.

    Documents

    Technical Specifications

    Applications Purified, converted DNA is of high-quality and well-suited for downstream processes, including library preparation for Next-generation sequencing, PCR amplification, etc.
    Conversion > 99.5%
    Elution Volume Shallow well: > 30 µl
    Deep-well: >15 µl
    Equipment Thermocycler with heated lid, swinging-bucket centrifuge with plate carriers.
    Input Samples containing between 50 pg to 2 µg of DNA. For optimal results, the amount of input DNA should be from 200 to 500 ng. The number of cells per standard treatment can range from 10-105 cells.
    Processing Time 4 hours
    Recovery > 80%
    Sample Source Blood, tissue, cells, FFPE, LCM-derived samples, purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc.
    Supplemental Info

    Resources

    http://www.zymoresearch.com/bisulfite-beginner-guide https://www.zymoresearch.com/bisulfite-primer-seeker

    Leaving the desulphonation buffer on the column longer than recommended will cause more degradation and subsequently result in lower yields.

    Bisulfite conversion will work regardless of context, so the kits are compatible with genomic DNA derived from plants and other species with high non-CpG methylation levels.

    ZymoTaq DNA Polymerase has been specifically designed for use in bisulfite amplification reactions. ZymoTaq is available as a stand-alone polymerase (E2001/E2002), PreMix (E2003/E2004), and qPCR PreMix (E2054/E2055).

    > 50 bp.

    For best results, keep the method of quantification consistent before and after bisulfite treatment:

    • If quantifying with a NanoDrop, use dsDNA settings (50 μg/ml for Ab260 = 1.0) before treatment and use RNA settings (40 μg/ml for Ab260 = 1.0) after treatment.
    • If quantifying with Qubit, use a dsDNA assay before treatment and use a ssDNA assay after treatment.
    Following bisulfite treatment of DNA, nonmethylated cytosine residues are converted into uracil. The recovered DNA is typically A, U, and T-rich. The original base-pairing no longer exists. Instead, the converted DNA is single stranded with limited non-specific base-pairing at room temperature. Therefore, traditional dsDNA methods of quantification will not accurately represent the amount of recovered bisulfite converted DNA.

    Following bisulfite treatment, DNA will be single stranded with limited non-specific base pairing at room temperature. To visualize, run the converted DNA on an agarose gel then chill the gel on ice or in an ice bath for 30 minutes. This will force enough base-pairing to allow intercalation of the ethidium bromide for the DNA to be visible. If using a Bioanalyzer or TapeStation instrument, use RNA kits and reagents to visualize the converted DNA.

    Poor conversion efficiency and low yields can be due to a variety of different experiment-specific conditions. Please contact Technical Support to discuss your specific experimental conditions and further troubleshoot with a product specialist.

    Converted DNA eluted in M-Elution Buffer can generally be stored at -20°C for 1-3 months. If longer term storage is necessary, we recommend storing at or below -70°C if possible. Bisulfite converted DNA is less stable than dsDNA; for best results, minimize freeze-thawing of converted DNA and use as soon as possible for downstream analysis.

    Cat # Name Size
    D5021-9 M-Digestion Buffer (2X) 15 ml
    D5021-8 M-Reaction Buffer 4ml
    D5021-7 M-Solubilization Buffer 18 ml
    D5006-3 M-Binding Buffer 125 ml
    D5007-6 M-Elution Buffer 8 ml
    D5006-2 M-Dilution Buffer-Gold 7 ml
    D5003-1 CT Conversion Reagent (96 Conversions) 1 Bottle
    D5002-5 M-Desulphonation Buffer 40 ml
    D5007-4 M-Wash Buffer 36 ml
    C2003 Elution Plate 2 Plates
    C2002 Collection Plate 2 Plates
    C2005 Conversion Plate w/ Cover Foil 2 Plates/Foils
    C2004 Zymo-Spin I-96 Plate 2 Plates
    C2001 Silicon-A Plate 2 Plates

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