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EZ-96 DNA Methylation-Lightning Kit

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Cat #	Name	Size
D5032	EZ-96 DNA Methylation-Lightning Kit (Shallow-Well)	2 x 96 Rxns.
D5033	EZ-96 DNA Methylation-Lightning Kit (Deep-Well)	2 x 96 Rxns.

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Highlights

Fastest 96-well bisulfite conversion kit for complete, high-throughput (96-well) bisulfite conversion of DNA for methylation analysis.
Ready-to-use conversion reagent is added directly to DNA.
High-yield, converted DNA is ideal for PCR, Methylation Specific PCR (MSP), arrays, library preps, Next-Generation sequencing, etc.

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Supplemental Info

Product Description

The EZ-96 DNA Methylation-Lightning Kit is a rapid, high-throughput (96-well) bisulfite conversion kit for DNA methylation analysis. The streamlined workflow features ready-to-use Lightning Conversion Reagent and a combined DNA denaturation and bisulfite conversion process. Desulphonation and clean-up of the converted DNA is performed using a unique 96-well spin-plate. With the Deep-Well Kit (Cat. D5033), samples can be eluted in as little as 15 µl. High yield, converted DNA is ideal for PCR, array, and Next-Generation sequencing, etc.

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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.
Conversion	> 99.5%
Elution Volume	Deep-well: ≥ 15 µl Shallow-well: ≥ 30 µl
Equipment	Thermocycler with heated lid, swinging-bucket centrifuge with plate carriers.
Input	100 pg - 2 µg of DNA.
Processing Time	1.5 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

Resources

Q1: Tips for bisulfite primer design?

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

Q2: 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).

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: 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.

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
D5032-1	Lightning Conversion Reagent	15 ml
D5031-5	L-Desulphonation Buffer	40 ml
D5006-3	M-Binding Buffer	125 ml
D5007-6	M-Elution Buffer	8 ml
D5007-4	M-Wash Buffer	36 ml
C2001	Silicon-A Plate	2 Plates
C2004	Zymo-Spin I-96 Plate	2 Plates
C2005	Conversion Plate w/ Cover Foil	2 Plates/Foils
C2002	Collection Plate	2 Plates
C2003	Elution Plate	2 Plates