The EZ-96 DNA Methylation-Direct MagPrep is a magnetic bead-based bisulfite conversion kit that allows simple and reliable DNA bisulfite conversion directly from blood, tissue, and cells without the prerequisite for DNA purification. The increased sensitivity of this magnetic bead-based bisulfite conversion kit makes it possible to amplify bisulfite-converted DNA from as few as 10 cells or 50 pg DNA. These innovations have been coupled to a magnetic bead-based clean-up for high-throughput methylation analysis. Desulphonation and clean-up of the converted DNA is performed while bound to the MagBinding Beads. This magnetic bead based bisulfite conversion kit has been designed to minimize template degradation, loss of DNA during treatment and clean-up, and to provide complete conversion of unmethylated cytosines. Recovered DNA is ideal for downstream analyses, including PCR amplification, endonuclease digestion, sequencing, microarrays, etc.
| Applications | Purified, converted DNA is of high-quality and well-suited for downstream processes, including library preparation for Next-Generation sequencing, PCR amplification, etc. |
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| Conversion | > 99.5% |
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| Elution Volume | ≥ 25 µl |
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| Equipment | Thermocycler with heated lid, heating element for 96-well plate, magnetic stand. |
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| 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. |
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| Processing Time | 4 hours |
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| Recovery | > 80% |
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| Sample Source | Blood, tissue, cells, FFPE, LCM-derived samples, purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc. |
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PRODUCT FAQ
Q1: Tips for bisulfite primer design?
Q2: Is incubation with Desulphonation Buffer for longer than 20 minutes recommended?
Leaving the desulphonation buffer on the column longer than recommended will cause more degradation and result in lower yields.
Q3: Does bisulfite conversion only occur in a CpG context?
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.
Q4: Which polymerase is recommended for amplification from bisulfite converted DNA?
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).
Q5: What is the minimum DNA size that can be recovered?
Q6: How to quantify / visualize converted DNA?
Following bisulfite treatment of genomic 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, it is single-stranded with limited non-specific base-pairing at room temperature. The absorption coefficient at 260 nm resembles that of RNA. Use a value of 40 μg/ml for Ab260 = 1.0 when determining the concentration of the recovered bisulfite-treated DNA. To visualize, run converted DNA on agarose gel then chill the gel on ice for 30 minutes. The expected smear will be between 100-1500bp.
Q7: What leads to poor conversion efficiency/ low yields?
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.
Q8: How long is bisulfite converted DNA stable at -20 °C?
Converted DNA should be eluted in M-Elution Buffer to keep the converted DNA stable for long term storage. If stored properly for long term (<-20C), the samples should last longer than a month. Minimize freeze/thawing to keep the bisulfite converted DNA stable.
KIT COMPONENTS
| Cat # | Name | Size |
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| C2003 | Elution Plate | 2 Plates |
| C2002 | Collection Plate | 2 Plates |
| C2005 | Conversion Plate w/ Cover Foil | 2 Plates/Foils |
| D5021-7 | M-Solubilization Buffer | 18 ml |
| D5021-8 | M-Reaction Buffer | 4ml |
| D5021-9 | M-Digestion Buffer (2X) | 15 ml |
| D5040-3 | M-Binding Buffer | 250 ml |
| D5040-5 | M-Desulphonation Buffer | 80 ml |
| D5040-4 | M-Wash Buffer | 72 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 |
| D4100-2-16 | MagBinding Beads | 16 ml |
| D4100-2-8 | MagBinding Beads | 8 ml |