The BisulFlash™ DNA Bisulfite Conversion Mag-96 Kit is a complete set of optimized reagents for fast, high throughput DNA bisulfite conversion. The kit uses a unique liquid conversion solution and magnetic binding bead clean-up process to quickly generate bisulfite-converted DNA for higher yields via a 96-well microplate. A magnetic bead based approach via microplates also allows for automation workflows. The kit has the following advantages:
- Fast - Complete the entire procedure in as little as 1 hour and 20 min for as many as 96 samples simultaneously, averaging just 50 seconds per sample.
- Convenient - Ready-to-use liquid conversion mix is simply added to the DNA samples directly, without need for pre-preparation of the conversion reagent.
- Streamlined - Concurrently processes the DNA denaturation and C to T conversion steps without the need for a separate DNA denaturation step.
- Complete Conversion - Completely converts unmethylated cytosine into uracil (>99.9%) with negligible inappropriate or error conversion of methylcytosine to thymine (<0.1%).
- Flexible - Choice of either (a) manual with one single reaction each time; or (b) high throughput with 96 reactions each time can be used, making the assay flexible.
- Robust - Simple, reliable, and consistent reaction conditions with an easy-to-follow protocol and high yield.
DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation. Gene/region-specific or genome-wide analysis of DNA methylation or 5-methylcytosine (5-mC) could provide valuable information for discovering epigenetic markers used for disease diagnosis, and potential targets used for therapeutics. By treating DNA with bisulfite, cytosine residues are deaminated to uracil while leaving 5-methylcytosine intact, so that methylated cytosine can be properly identified via PCR, array, bisulfite sequencing, or next generation sequencing.
Principle & Procedure
This kit contains all reagents required for a fast bisulfite conversion in a high throughput format. With the unique conversion mix solution, DNA denaturation status is sustained throughout the entire bisulfite conversion process, thereby enabling full conversion of unmethylated cytosine to uracil. Desulphonation and clean-up of the converted DNA is performed using magnetic bead-based binding and separation in a 96-well microplate. We recommend using the EpiMag HT Magnetic Separator. High yield, converted DNA can be obtained and used for various downstream applications including PCR and next generation sequencing.
Starting materials may include various tissue or cell samples such as: cultured cells from a flask or microplate, microdissection sample, paraffin-embedded tissue, plasma/serum sample, and body fluid sample, etc. The amount of DNA for each reaction can be 0.1 to 1 ug. For an optimal reaction, the input DNA amount should be 100 ng. If small amounts (e.g., <10 ng) of starting DNA are used, the number of PCR cycles should be greater than 45.
Fig. 1. Schematic procedure of the BisulFlash DNA Bisulfite Conversion Mag-96 Kit .
Fig. 2. Different amounts of human genomic DNA were converted using the BisulFlash DNA Bisulfite Conversion Mag-96 Kit. Converted DNA was amplified using Methylamp™ MS-qPCR Fast Kit (Cat. # P-1028).
Fig. 3. High accuracy of DNA conversion is achieved by the BisulFlash DNA Bisulfite Conversion Mag-96 Kit . 100 ng of genomic DNA methylated in all CpG sites by DNA methylases was treated with the kit followed by real time qPCR amplification using primers for multiple promoters containing numerous CpG sites and then directly sequenced. 100% C at non-CpG sites are converted to T and 100% C at CpG sites remain as C.