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EZ DNA Methylation Kit

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Cat #	Name	Size
D5001	EZ DNA Methylation Kit	50 Rxns.
D5002	EZ DNA Methylation Kit	200 Rxns.

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Highlights

Streamlined, proven procedure for bisulfite conversion of DNA.
Desulphonation and recovery of bisulfite-treated DNA with a spin column.
Recovered DNA is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.

Documents

Product Description

The EZ DNA Methylation Kit features a simplified procedure that streamlines bisulfite treatment of DNA. This kit is the original bisulfite conversion kit from Zymo Research. The EZ DNA Methylation Kit is based on the three-step reaction that takes place between cytosine and sodium bisulfite during which cytosine is converted into uracil. Innovative desulphonation technologies eliminate 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 library preparation for Next-generation sequencing, PCR amplification, endonuclease digestion, sequencing, microarrays, etc. The EZ DNA Methylation Kit is recommended for use with 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. This kit is recommended for use with Illumina Infinium MethylationEPIC BeadChip array.
Conversion	>99%
Elution Volume	≥10 µl
Equipment	Microcentrifuge and thermocycler with heated lid
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.

Resources

Q1: 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.

Q2: Tips for bisulfite primer design?

https://www.zymoresearch.com/pages/bisulfite-beginner-guide
https://www.zymoresearch.com/pages/bisulfite-primer-seeker

Q3: 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).

Q4: 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.

Q5: What is the minimum DNA size that can be recovered?

> 50 bp.

Q6: 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.

Q7: 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.

Q8: 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.

Q9: 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
C1004-50	Zymo-Spin IC Columns	50 Pack
C1001-50	Collection Tubes	50 Pack
D5001-4	M-Wash Buffer	6 ml
D5002-4	M-Wash Buffer	24 ml
D5002-3	M-Binding Buffer	80 ml
D5001-5	M-Desulphonation Buffer	10 ml
D5002-5	M-Desulphonation Buffer	40 ml
D5001-6	M-Elution Buffer	1 ml
D5002-6	M-Elution Buffer	4 ml
D5001-3	M-Binding Buffer	20 ml
D5001-2	M-Dilution Buffer	1.3 ml
D5002-2	M-Dilution Buffer	5.2 ml
D5001-1	CT Conversion Reagent	1 tube for 10 Conversions