EZ DNA Methylation-Gold™ Kit

Complete bisulfite conversion of GC-rich DNA in less than 3 hours.
A coupled heat denaturation/conversion reaction step steamlines the conversion of non-methylated cytosines into uracil.
Desulfonation and recovery of bisulfite-treated DNA with a spin column.
Recovered DNA is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.

Product Size Catalog # Price Qty
EZ DNA Methylation-Gold™ Kit 50 Rxns D5005
$148.00
EZ DNA Methylation-Gold™ Kit 200 Rxns D5006
$497.00

About EZ DNA Methylation-Gold™ Kit

The EZ DNA Methylation-Gold™ Kit is a refinement of our popular EZ DNA Methylation™ kits. These products consolidate DNA denaturation and bisulfite conversion processes into one step, leading to a much faster bisulfite conversion. This is accomplished using temperature denaturation to complement chemical denaturation in the previous protocol. Also, this kit have been streamlined for high yield recovery of DNA following bisulfite treatment. Recovered bisulfite-converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion, sequencing, microarrays, etc.

Learn More About Bisulfite Conversion Click to see which kit is right for you


Format Spin Column
DNA Recovery > 75%
Processing Time 3 hrs
Equipment Microcentrifuge
Binding Capacity 5 µg/prep
Elution Volume ≥ 10 µl
Conversion Efficiency > 99%
Input Plasmid, genomic, and endonuclease digested DNAs.

DNA sequencing results after bisulfite treatment. DNA with methylated CmpG (at nucleotide position #5) was processed using the EZ DNA Methylation-Gold™ Kit. The recovered DNA was amplified by PCR and then sequenced directly. The methylated cytosine at position #5 remained intact while the unmethylated cytosines (i.e., positions #7, 9, 11, 14 and 15) were completely converted into uracil following bisulfite treatment and detected as thymine following PCR.

 

 

 

 

 

Step 1

Add 130 µl of the CT Conversion Reagent to 20 µl of your DNA sample in a PCR tube.  If the volume of the DNA sample is less than 20 µl, make up the difference with water.  Mix the sample by flicking the tube or pipetting the sample up and down, then centrifuge the liquid to the bottom of the tube. 

Step 2

Place the sample tube in a thermal cycler and perform the following steps*:

1.   98°C for 10 minutes

2.   64°C for 2.5 hours

3.     4°C storage up to 20 hours

*For some samples, alternative parameters may yield improved results (see Appendix).  If you have been using this kit with good results using different reaction conditions than described above, you can continue using those same conditions.

Step 3

Add 600 µl of M-Binding Buffer to a Zymo-Spin™ IC Column and place the column into a provided Collection Tube.

Step 4

Load the sample (from Step 2) into the Zymo-Spin™ IC Column containing the M-Binding Buffer.  Close the cap and mix by inverting the column several times.

Step 5

Centrifuge at full speed (≥10,000 x g) for 30 seconds.  Discard the flow-through.

Step 6

Add 100 µl of M-Wash Buffer to the column.  Centrifuge at full speed for 30 seconds.

Step 7

Add 200 µl of M-Desulphonation Buffer to the column and let stand at room temperature (20-30°C) for 15-20 minutes.  After the incubation, centrifuge at full speed for 30 seconds.

Step 8

Add 200 µl of M-Wash Buffer to the column.  Centrifuge at full speed for 30 seconds.  Add another 200 µl of M-Wash Buffer and centrifuge for an additional 30 seconds.

Step 9

Place the column into a 1.5 ml microcentrifuge tube.  Add 10 µl of M-Elution Buffer directly to the column matrix.  Centrifuge for 30 seconds at full speed to elute the DNA.

The DNA is ready for immediate analysis or can be stored at or below -20°C for later use.  For long term storage, store at or below -70°C.  We recommend using 1-4 µl of eluted DNA for each PCR, however, up to 10 µl can be used if necessary.  The elution volume can be < 10 µl depending on the requirements of your experiments, but small elution volumes will yield more concentrated DNA.

For specific notes and additional information, please see the product protocol PDF.
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Featured Citations

Researchers from the National Institute of Plant Genome Research in New Delhi used the EZ DNA Methylation-Gold™ Kit to identify the level of cytosine methylation in genomic DNA from Tomato leaf curl New Delhi virus. Through DNA methylation-specific RNA silencing, the researchers aimed to target the viral genomic regions of this virus to potentially determine an alternate defense mechanism for generating transgenic plants to prevent yield loss.
Sahu P et al. Post-transcriptional and Epigenetic Arms of RNA Silencing: A Defense Machinery of Naturally Tolerant Tomato Plant Against Tomato Leaf Curl New Delhi Virus. Plant Mol Biol Rep (2014) 32:1015-1029
The EZ DNA Methylation-Gold™ Kit was used to bisulfite convert DNA extracted from Thy-1 (+) and Thy-1 (-) lung fibroblasts and tissue. Data from methylation-specific PCR and bisulfite sequencing enabled researchers to show that epigenetic regulation of Thy-1 could be a novel and potentially reversible mechanism in fibrosis that may offer the possibility of new therapeutic options.
Researchers from China analyzed the role of DNA methylation in the life cycle of a parasitic nematode, Trichinella spiralis and characterized the methylome of three life-cycles states in this organism. Genomic DNA of T. spiralis muscle larvae, adults, and new-born larvae was isolated and methylated cytosine levels were determined by MethylC-seq.
Genomic DNA was extracted from paraffin-embedded (FFPE) renal cancer tissue blocks and was bisulfite converted using EZ DNA Methylation-Gold Kit. The authors found CST6 and LAD1 methylation to strongly associate with progression-free and overall survival of patients undergoing VEGF-targeted therapy for metastasized renal cell cancer, suggesting these sites as candidate biomarkers for predicting disease progression and response to therapy.
European scientists used the EZ DNA Methylation-Gold Kit and pyrosequencing to verify results from a DNA methylation array. They found that anaplastic Pleomorphic xanthoastrocytoma (PXA) has increasing DNA promoter hypermethylation compared with grade II samples.
Genomic DNA was extracted from T cells from blood and DNA methylation was analyzed by Methylated DNA immunoprecipitation (MeDIP) and Microarray Hybridization. Next, the researchers validated the MeDIP data by Illumina Infinium HumanMethylation450 arrays and by pyrosequencing. This study presents preliminary evidence for genome-wide variation in promoter DNA methylation associated with chronic physical aggression.
DNA was extracted from cervical cytology samples and the presence of HPV16 was confirmed by PCR. DNA was then bisulfite converted using EZ DNA Methylation-Gold kit, amplified, and sequenced. The authors noted an increase in HPV16 biomarker methylation with increasing histologic severity.
Researchers from Siberian Branch of the Russian Academy of Sciences and Charite-Universitatsmedizin Berlin evaluated three commercially available bisulfite modification kits using cell-free circulating DNAs (cfcDNA) in blood. They showed that the conversion efficiency of EZ DNA Methylation-Gold™ Kit is close to 99% and this kit also provided the highest recovery (> 86%) regardless of the initial DNA input. This paper suggests that the EZ DNA Methylation-Gold™ Kit is the most appropriate tool for studying bisulfite modification of low concentration and fragmented cfcDNA.
Researchers used the EZ DNA Methylation-Gold kit from Zymo Research to investigate the role of the antiviral protein Daxx on the methylation of retroviral DNA. They found that Daxx was responsible for promoting methylation of the viral genome, and it may play a direct role in the epigenetic silencing of viral gene expression because Daxx was observed to associate with both DNA methyltransferase enzymes and viral DNA.
To understand the basis of inheritance of DNA methylation patterns from gametes to offspring in zebrafish, Next-Gen sequencing libraries were constructed from genomic DNA of sperm, oocytes, and embryos from different stages. The libraries were bisulfite converted using the EZ DNA Methylation-Gold kit from Zymo Research. Sequencing results showed early embryos inherit DNA methylomes from sperm rather than oocytes.
The EZ DNA Methylation-Gold™ Kit was used to investigate RNA-dependent RNA polymerase 6 (RDR6)-mediated RNA-directed DNA methylation of transposable elements (TEs) in Arabidopsis thaliana. The authors identified RDR6-specific small interfering RNAs (siRNAs) that were expressed from transcriptionally active TEs and demonstrated that these siRNAs were responsible for initiating the silencing of active transposable elements by promoting DNA methylation through an RNA-dependent mechanism.
The EZ DNA Methylation – GoldTM kit was used for the bisulfite treatment of DNA from tumor tissues of Dutch and Chinese patients with Lynch syndrome. Data from methylation-specific PCR and pyrosequencing of the bisulfite-converted DNA suggested that the absence of polyadenylation in an expressed gene, TACSTD1, may result in epigenetic inactivation of its downstream neighbor, MSH2, possibly contributing to disease.
Genomic DNA from HeLa cells was treated with sodium bisulfite and desulfonated using the EZ DNA Methylation-Gold Kit. Bisulfite-converted DNA was used for library preparation and sequencing to analyze the DNA methylation status of the intergenic spacer region of the ribosomal DNA locus.
Honeybee genomic DNA was bisulfite converted with the EZ DNA Methylation-GoldTM kit. The converted DNA was used for CHARM analysis and whole genome bisulfite sequencing to compare DNA methylation levels between subclasses of worker bees.
To study the effect of curcumin on DNA methylation in colorectal cancer (CRC) cells, genomic DNA from three CRC cell lines was bisulfite converted using the EZ DNA Methylation – GoldTM kit. Data gathered using various assays, such as microarrays, pyrosequencing, and real-time PCR, suggest that treatment with curcumin induces gene- and cell-line-specific methylation changes and that these changes have a direct impact on the expression of genes involved in major biological processes.

“This protocol allows for bisulfite conversion in about 3 hours. This fast conversion is particularly relevant to our experiments, which allows us to PCR amplify and clone the products in 1 day, saving time without loss of quality.”
Joseph M. (Sanford-Burnham Medical Research Institute)
“It makes gene specific DNA methylation analysis very simple and anyone with modest technical background can easily perform the experiment using this kit.”
Murali B. (Centre for DNA Fingerprinting and Diagnostics)
“It works better than competitor's kits, with a much quicker protocol.”
Esteban C.
“The kit is easy to follow and produces efficient, high-quality results.”
Daniel S. (Wake Forest Baptist Health)
“It is very easy to use and the cleanup steps after the bisulfite treatment are fast.”
Marco M. (UCLA)
Click here to submit your review of the EZ DNA Methylation-Gold™ Kit.

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