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EZ DNA Methylation-Lightning™ Kit

Fastest method for complete 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, MSP, array, bisulfite and Next-Gen sequencing.

Product Size Catalog # Price Qty
EZ DNA Methylation-Lightning™ Kit 10 rxns D5030T
EZ DNA Methylation-Lightning™ Kit 50 rxns D5030
EZ DNA Methylation-Lightning™ Kit 200 rxns D5031

About EZ DNA Methylation-Lightning™ Kit

The EZ DNA Methylation-Lightning™ Kit features rapid and reliable bisulfite treatment and conversion of DNA for methylation analysis. Key to the fast workflow is the ready-to-use Lightning Conversion Reagent. No preparation is necessary, simply add this unique reagent to a DNA sample, wait about an hour, and let the reaction proceed to completion. DNA denaturation and bisulfite conversion processes are combined with added heat to facilitate rapid denaturation. Desulphonation and clean-up of the converted DNA is performed using a unique low-elution spin column. High yield, converted DNA is ideal for PCR, array, bisulfite and next generation sequencing, etc.

Learn More About Bisulfite Conversion Click to see which kit is right for you

DNA Recovery >80%
Conversion Efficiency >99.5% of non-methylated cytosine residues are converted to uracil; >99.5% protection of methylated cytosines.
Optimal DNA Input Samples containing between 100 pg to 2 µg of DNA. For optimal results, the amount of input DNA should be from 200 to 500 ng.

DNA sequencing results following bisulfite treatment. DNA with methylated C at nucleotide position #5 was processed using the EZ DNA Methylation™ Kit. The recovered DNA was amplified by PCR and then sequenced directly. The methylated cytosine at position #5 remains intact while the unmethylated cytosines at positions #7, 9, 11, 14 and 15 are completely converted into uracil following bisulfite treatment (detected as thymine following PCR).

Methylation Plot From Reduced Representation Bisulfite Sequencing (RRBS). Data shows the relative percentage of methylation at individual CpG sites in mouse DNA. Methylation percentage is shown across a ~3 Mb region of mouse chromosome 19. Bisulfite sequencing libraries were prepared using mouse genomic DNA prepped with the Genomic Clean & Concentrator™ (D4010, D4011 – Zymo Research) and bisulfite converted using EZ DNA Methylation™ technology prior to Next-Gen sequencing.

Zymo-Spin™ IC Design Characteristics. The image above shows the unique design of the Zymo column that facilitates extremely small elution volumes (≥10 µl) without buffer carryover. This is unlike other columns that can retain liquid (binding/wash buffer residue) leading to carryover into the eluate.

Step 1

Add 130 µl of Lightning Conversion Reagent to 20 µl of a DNA sample in a PCR tube.  Mix, then centrifuge briefly to ensure there are no droplets in the cap or sides of the tube.

Note: If the volume of DNA is less than 20 µl, compensate with water.

Step 2

Place the PCR tube in a thermal cycler and perform the following steps:

1.    98°C  for 8 minutes

2.    54°C  for 60 minutes

3.      4°C  storage for up to 20 hours 

Note: The 4°C storage step is optional.

Step 3

Add 600 µl of M-Binding Buffer to a Zymo-Spin™ IC Column and place the column into a provided Collection Tube.

Step 4

Load the sample (from Step 2) into the Zymo-Spin™ IC Column containing the M-Binding Buffer.  Close the cap and mix by inverting the column several times.

Step 5

Centrifuge at full speed (≥10,000 x g) for 30 seconds.  Discard the flow-through.

Step 6

Add 100 µl of M-Wash Buffer to the column.  Centrifuge at full speed for 30 seconds.

Step 7

Add 200 µl of L-Desulphonation Buffer to the column and let stand at room temperature (20-30°C) for 15-20 minutes.  After the incubation, centrifuge at full speed for 30 seconds.

Step 8

Add 200 µl of M-Wash Buffer to the column.  Centrifuge at full speed for 30 seconds.  Repeat this wash step.

Step 9

Place the column into a 1.5 ml microcentrifuge tube and add 10 µl of M-Elution Buffer directly to the column matrix.  Centrifuge for 30 seconds at full speed to elute the DNA.

The DNA is ready for immediate analysis or can be stored at or below -20°C for later use.  For long term storage, store at or below -70°C.  We recommend using 1-4 µl of eluted DNA for each PCR, however, up to 10 µl can be used if necessary.  The elution volume can be < 10 µl depending on the requirements of your experiments, but small elution volumes will yield higher DNA concentrations.

For specific notes and additional information, please see the product protocol PDF.
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Featured Citations

Researchers studying circulating unmethylated and methylated preproinsulin (INS) DNA in type 1 diabetes (T1D) sought to test the differences between subjects with new-onset T1D and controls. DNA was extracted from serum using the ZR Serum DNA Kit™ and bisulfite converted with the EZ DNA Methylation Kit™ or the EZ DNA Methylation-Lightning Kit™. Droplet digital PCR was used to measure unmethylated and methylated serum levels. The data demonstrated that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. The use of methylation levels as potential biomarkers for diabetes may prove to be a novel tool in the near future.
The EZ DNA Methylation-Lightning™ Kit was used to bisulfite convert DNA from prefrontal cortex samples of patients with psychiatric diseases. The researchers then used a recently developed bisulfite padlock probe-based approach for ultra-deep mapping of modified cytosines in patients affected or unaffected by major psychosis. Their findings revealed significant epigenetic differences between patients and controls and also identified interesting epigenetic age effects.
In order to investigate why endogenes are less susceptible to RNA silencing than transgenes, researchers from Germany used the EZ DNA Methylation-Lightning™ Kit to bisulfite convert DNA from locally and systemically silenced transgenic plant leaves. After amplifying the bisulfite-converted DNA using ZymoTaq™ polymerase and bisulfite sequencing, the researchers were able to conclude that cDNA transgenes are prone to post-transcriptional gene silencing and RNA-directed DNA methylation while endogene-resembling transgenes are resistant to systemic silencing and DNA methylation.
DNA from FACS-purified prospermatogonia (PSG) was bisulfite converted using the EZ DNA Methylation-Lightning™ Kit. Genome-wide single-base-resolution DNA methylome analysis revealed epigenetic deficiencies in mutant germ cells specifically cell types expressing Dnmt3L. The data also implies that genomes of germ cells may have the highest overall levels of DNA methylation which is proposed to be a consequence of Dnmt3L.
Cerebellar cortex DNA from both Autism spectrum disorder (ASD) and healthy individuals were bisulfite converted using the EZ DNA Methylation-Lightning Kit in parallel to immunoprecipitation (with either 5-hmC or 5-mC antibodies). Next, 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) were measured with pyrosequencing. The researchers reported that an increase of MeCP2 binding at GAD1 and RELN promoters is associated with increased levels of 5-hmC in ASD cerebellum.
The EZ DNA Methylation-Lightning Kit to bisulfite convert DNA extracted from cheek cells or preserved kidney tissues from dogs with clinical cases of moderate to severe renal dysplasia. It was found that wild-type alleles of the Cox-2 promoter are not methylated, however, allelic variants of the Cox-2 promoter that result in clinical manifestation of renal dysplasia have aberrant methylation.
Researchers from New York evaluated the DNA methylation patterns between individuals with Autism Spectrum Disorders (ASD) and typical developing (TD) controls. Using the EZ DNA Methylation-Lightning Kit, DNA extracted from the homogeneous ectodermal cells type was bisulfite converted and analyzed using a quantitative genome-wide DNA methylation assay. DNA methylation patterns in individuals with ASD were shown to be dysregulated compared to the typical developing individuals.
To determine the role Tet1 may have in activating meiotic genes, whole-genome bisulfite sequencing libraries were generated to compare the methylation levels of wild-type and Tet1-mutated mouse primordial germ cells (PGCs). The authors found that libraries bisulfite converted using the EZ DNA Methylation-Lightning™ Kit from Zymo Research were fully converted and post-mapping filtering was not required, whereas libraries processed with the Sigma Imprint Kit had some poorly converted reads, which required post-mapping filtering. The results of the findings showed Tet1 is not responsible for global demethylation in PGCs but is involved in specific meiotic gene activation via DNA demethylation.

“The conversion reagent no longer has to be made up from individual components and dissolved - it is supplied ready-made. The conversion reagent can be stored at room temperature - there is no need to use up all 10 uses in one month. This makes it more flexible, and avoids wastage. Shorter protocol saves time.”
Jeremy B. (AgResearch)
“Results were as good as with the formerly used EZ DNA Methylation Gold Kit but significantly reduced total time for analyses. More flexibility due to the possibility of up to 20h storage of the samples after bisulfite conversion before the cleaning procedure especially for part-time employees.”
Sabrina S. (UK-Aachen)
“Fast conversion step, easy clean-up with very good recovery from the columns”
Evelyn H. (Salzburger Universitätklinikum)
“Easy to use and its compatibility with Next generation sequencing protocols (RRBS)”
Satish S. (Institute of Genomics and Integrative Biology)
“I like the fact that I don't have to wait more than an hour after the incubation for the bisulfite conversion.”
Marco M. (UCLA)
“It’s robust and quick, one can produce good results in no time.”
Ahmed A. (Uppsala Universitet)
Click here to submit your review of the EZ DNA Methylation-Lightning™ Kit.

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