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DNA Bisulfite Conversion

bisulfite conversion and PCR amplification of methylated DNABisulfite conversion is a popular technique used to study DNA methylation that involves converting cytosine to uracil while leaving 5-methylcytosine (5-mC) intact. Bisulfite and sodium bisulfite based methods are common approaches for preparing genomic DNA for gene-specific DNA methylation analysis.

Bisulfite conversion is considered the “gold standard” for analyzing DNA methylation and can be followed by downstream applications such as PCR or sequencing. Unmethylated cytosine residues are deaminated to uracil and methylated cytosine (5-mC) residues remain unaffected, enabling PCR amplification to recognize uracils as thymines and 5-mC or 5-hmC as cytosines. This allows researchers to differentiate between methylated and unmethylated cytosine residues, offering single-nucleotide resolution information about the methylated areas of DNA. Complete conversion is necessary for a successful DNA methylation study, along with reducing the amount of DNA degradation that will occur as a result of the typically harsh chemical reaction.

chemical structure showing bisulfite conversion of cytosine to uracilPopular downstream methods for analyzing DNA methylation on a gene-specific basis following bisulfite treatment include bisulfite sequencing, methylation specific PCR (MS-PCR), and methylation-based microarrays. Considering your downstream application can help in determining which bisulfite conversion method is ideal, especially due to the variation in post-conversion fragment size. The BisulFlash DNA Modification Kit is geared specifically toward real-time MSP whereas the best-selling Methylamp DNA Modification Kit is designed to be compatible with Illumina workflows and next-generation sequencing (NGS). If a quicker and easier bisulfite conversion alternative is desired prior to sequencing, the BisulFlash DNA Bisulfite Conversion Easy Kit can be completed in just 60 minutes and uses a liquid bisulfite conversion reagent for convenience.

Beginning with high quality DNA is critical, especially because the acidic nature of the conversion process will fragment the DNA. For samples containing low amounts of DNA, the Methylamp Whole Cell Bisulfite Modification Kit is recommended, as it can directly convert from cells, tissues, blood, and other starting materials. When handling large scale bisulfite conversion experiments, high-throughput options are critical in order to save time and reduce cost. In this case, the BisulFlash DNA Bisulfite Conversion Mag-96 Kit is specifically streamlined for high-throughput and uses a magnetic bead-based 96-well format. For a detailed comparison of bisulfite conversion kits, see the table below.

 

Methylamp DNA Modification KitCat #P-1001

BisulFlash DNA Bisulfite Conversion Easy KitCat #P-1054

BisulFlash DNA Modification KitCat #P-1026

Methylamp Whole Cell Bisulfite Modification KitCat #P-1016

BisulFlash DNA Bisulfite Conversion Mag-96 KitCat #P-1050

  More DetailsMore DetailsMore DetailsMore DetailsMore Details
Codename"Classic""Easy""Fast""Direct""High Throughput"
PurposeChemical-based DNA bisulfite conversionSimplified heat-based DNA bisulfite conversionFast heat-based DNA bisulfite conversionDirectly convert from cells, tissues, saliva, urine, or bloodMagnetic bead-based 96-well format bisulfite conversion
Key Points
  • Illumina workflow compatible
  • Most robust and reliable
  • Ideal for bisulfite sequencing, NGS, and various MSP
  • Illumina workflow compatible
  • Denaturation by thermalcycler
  • Liquid bisulfite conversion reagent
  • Extremely fast
  • Ideal for real-time MSP
  • Optional sequencing protocol
  • Eliminates separate DNA isolation step
  • Ideal for samples containing low quantities of DNA
  • Illumina workflow compatible
  • Streamlined high throughput process
  • Designed for large scale or automated conversions
Starting MaterialDNADNADNAcells, tissue, bloodDNA
Lowest Input Amount50 pg100 pg200 pg100 cells, 1 µl blood< 10 ng
Total Protocol Time< 2 hours1 hour30 minutes< 3 hours< 1 hour 20 min
Elution Volume8-20 µl8-20 µl10-20 µl8-20 µl20 µl
Conversion Efficiency99.9%99.9%99.9%99.9%99.9%
Desulfonation/Clean-Up
Recovery>75%>75%>75%>85%>85%
Post-Conversion DNA Fragment Size200-2000 bp; Avg. Peak of 800 bp200-2000 bp; Avg. Peak of 800 bp100-400 bp; Avg. Peak of 250 bp200-2000 bp; Avg. Peak of 800 bp200-2000 bp; Avg. Peak of 800 bp
Vessel FormatSpin ColumnSpin ColumnSpin ColumnSpin Column96-Well Plate
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