Highlights
- High-throughput (96-well) bisulfite conversion of DNA in less than 3 hours. A coupled heat denaturation/conversion reaction step streamlines the conversion of non-methylated cytosines into uracil.
- Desulphonation and recovery of bisulfite-treated DNA with a 96-well plate.
- Recovered DNA is ideal for downstream analyses such as PCR, endonuclease digestion, sequencing, microarrays, etc.
Description
The EZ-96 DNA Methylation-Gold Kit allows high-throughput (96-well spin-plate) conversion of unmethylated cytosines into uracil in less than 3 hours. Our optimized bisulfite chemistry combines heat denaturation and bisulfite conversion of input DNA to reduce incubation time and ensure conversion rates >99%. Recovered bisulfite-converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion, sequencing, microarrays, etc.
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. Catalog # D5004 is recommended for use with Illumina Infinium MethylationEPIC BeadChip array. |
|---|---|
| Conversion | >99% |
| Elution Volume | ≥ 15 µl for Deep-well ≥ 30 µl for Shallow-well |
| Equipment | Thermocycler with heated lid, swinging-bucket centrifuge with plate carriers |
| Input | 500 pg – 2 µg of DNA. |
| Processing Time | 3 hours |
| Recovery | >75% |
| Sample Source | Purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc. DNA should be high-quality and RNA-free. |
Product FAQ
Q1: Tips for bisulfite primer design?
https://www.zymoresearch.com/pages/bisulfite-beginner-guide
https://www.zymoresearch.com/pages/bisulfite-primer-seeker
Q2: Is an 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 subsequently 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?
> 50 bp.
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.
Citations
Reviews
“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)
Kit Components
| Cat # | Name | Size |
|---|---|---|
| D5007-4 | M-Wash Buffer | 36 ml |
| D5002-5 | M-Desulphonation Buffer | 40 ml |
| D5007-6 | M-Elution Buffer | 8 ml |
| D5003-1 | CT Conversion Reagent (96 Conversions) | 1 Bottle |
| D5006-2 | M-Dilution Buffer-Gold | 7 ml |
| D5006-3 | M-Binding Buffer | 125 ml |
| D5006-6 | M-Dissolving Buffer | 1.2 ml |
| C2001 | Silicon-A Plate | 2 Plates |
| C2002 | Collection Plate | 2 Plates |
| C2003 | Elution Plate | 2 Plates |
| C2004 | Zymo-Spin I-96 Plate | 2 Plates |
| C2005 | Conversion Plate w/ Cover Foil | 2 Plates/Foils |
EZ-96 DNA Methylation-Gold Kit
D5008 / D5007
| Cat # | Name | Size |
|---|
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