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EZ-96 DNA Methylation Kit
D5004 / D5003
EZ-96 DNA Methylation Kit
- 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.
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.
|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.|
|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|
|Sample Source||Purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc. DNA should be high-quality and RNA-free.|
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 converted DNA?
For best results, keep the method of quantification consistent before and after bisulfite treatment:
- If quantifying with a NanoDrop, use dsDNA settings (50 μg/ml for Ab260 = 1.0) before treatment and use RNA settings (40 μg/ml for Ab260 = 1.0) after treatment.
- If quantifying with Qubit, use a dsDNA assay before treatment and use a ssDNA assay after treatment.
Q3: How to visualize converted DNA?
Following bisulfite treatment, DNA will be single stranded with limited non-specific base pairing at room temperature. To visualize, run the converted DNA on an agarose gel then chill the gel on ice or in an ice bath for 30 minutes. This will force enough base-pairing to allow intercalation of the ethidium bromide for the DNA to be visible. If using a Bioanalyzer or TapeStation instrument, use RNA kits and reagents to visualize the converted DNA.
Q4: What is the minimum DNA size that can be recovered?
> 50 bp.
Q5: How long is bisulfite converted DNA stable at -20 °C?
Converted DNA eluted in M-Elution Buffer can generally be stored at -20°C for 1-3 months. If longer term storage is necessary, we recommend storing at or below -70°C if possible. Bisulfite converted DNA is less stable than dsDNA; for best results, minimize freeze-thawing of converted DNA and use as soon as possible for downstream analysis.
Q6: 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.
Q7: 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.
Q8: 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).
Q9: Tips for bisulfite primer design?
|D5002-2||M-Dilution Buffer||5.2 ml|
|D5007-6||M-Elution Buffer||8 ml|
|D5003-1||CT Conversion Reagent (96 Conversions)||1 Bottle|
|D5002-5||M-Desulphonation Buffer||40 ml|
|D5002-3||M-Binding Buffer||80 ml|
|D5007-4||M-Wash Buffer||36 ml|
|C2003||Elution Plate||2 Plates|
|C2002||Collection Plate||2 Plates|
|C2005||Conversion Plate w/ Cover Foil||2 Plates/Foils|
|C2001||Silicon-A Plate||2 Plates|
|C2004||Zymo-Spin I-96 Plate||2 Plates|