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EZ-96 DNA Methylation Kit

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Cat #	Name	Size
D5004	EZ-96 DNA Methylation Kit (Deep-Well)	2 x 96 Rxns
D5003	EZ-96 DNA Methylation Kit (Shallow-Well)	2 x 96 Rxns

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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.

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Supplemental Info

Product 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.

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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.
Supplemental Info	Bisulfite Beginner Guide Controls for DNA Methylation Assays Bisulfite Conversion for Illumina Methylation Assays Bisulfite Primer Design Tool Link

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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.

Following bisulfite treatment of 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, the converted DNA is single stranded with limited non-specific base-pairing at room temperature. Therefore, traditional dsDNA methods of quantification will not accurately represent the amount of recovered bisulfite converted DNA.

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.

Cat #	Name	Size
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