Highlights
- Streamlined, proven procedure for bisulfite conversion of DNA.
- Desulphonation and recovery of bisulfite-treated DNA with a 96-well spin-column plate.
- Recovered DNA is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.
Description
The EZ-96 DNA Methylation Kits are bisulfite conversion kits that feature a high-throughput (96-well spin-plate), simplified procedure that streamlines bisulfite conversion of DNA. Cytosines undergo a three-step reaction with sodium bisulfite during which the cytosine is converted into uracil. The innovative in-column desulphonation technology eliminates otherwise cumbersome precipitations. The kit is designed to reduce template degradation and minimize DNA loss during treatment and clean-up, while ensuring complete conversion of the DNA. Purified, converted DNA is ideal for downstream analyses including PCR amplification, endonuclease digestion, sequencing, microarrays, etc.
Note: Catalog # D5004 is recommended for use with the Illumina Infinium MethylationEPIC BeadChip array.
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 | 12-16 hours |
| Recovery | >80% |
| Sample Source | Purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc. DNA should be high-quality and RNA-free. |
Product FAQ
Q1: 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.
Q2: 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.
Q3: What is the minimum DNA size that can be recovered?
> 50 bp.
Q4: 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.
Q5: 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.
Q6: 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.
Q7: 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).
Q8: Tips for bisulfite primer design?
Citations
Kit Components
| Cat # | Name | Size |
|---|---|---|
| D5002-2 | M-Dilution Buffer | 5.2 ml |
| D5002-3 | M-Binding Buffer | 80 ml |
| 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 |
| C2001 | Silicon-A Plate | 2 Plates |
| C2002 | Collection Plate | 2 Plates |
| C2003 | Elution Plate | 2 Plates |
| C2005 | Conversion Plate w/ Cover Foil | 2 Plates/Foils |
