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EZ-96 DNA Methylation-Lightning™ Kit

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

Product Size Catalog # Price Qty
EZ-96 DNA Methylation-Lightning™ Kit (Shallow-Well) 2x96 rxns D5032
EZ-96 DNA Methylation-Lightning™ Kit (Deep-Well) 2x96 rxns D5033

About EZ-96 DNA Methylation-Lightning™ Kit

The EZ-96 DNA Methylation-Lightning™ Kit features high-throughput (96-well) 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 96-well spin-plate. 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.

Step 1

Add 130 µl of Lightning Conversion Reagent to 20 µl of a DNA sample in a Conversion Plate.  Mix the samples by pipetting up and down.

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

Step 2

Seal the plate with the provided film.  Transfer the Conversion Plate to 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 400 µl of M-Binding Buffer to the wells of a Silicon-A™ Binding Plate mounted on a Collection Plate.  

Step 4

Transfer the samples from the Conversion Plate (Step 2) to the wells of the Silicon-A™ Binding Plate.  Mix by pipetting up and down.

Step 5

Centrifuge at ≥ 3,000 x g (5,000 x g max.) for 5 minutes.  Discard the flow-through.

Step 6

Add 400 µl of M-Wash Buffer to each well of the plate.  Centrifuge at ≥ 3,000 x g for 5 minutes.

Step 7

Add 200 µl of M-Desulphonation Buffer to each well and allow the plate to stand at room temperature (20-30°C) for 15-20 minutes.  After the incubation, centrifuge at ≥ 3,000 x g for 5 minutes.  Discard the flow-through.

Step 8

Add 400 µl of M-Wash Buffer to each well of the plate.  Centrifuge at ≥ 3,000 x g for 5 minutes.  Discard the flow-through.  Add another 400 µl of M-Wash Buffer and centrifuge for 10 minutes.

Step 9

Place the Silicon-A™ Binding Plate onto an Elution Plate.  Add 30 µl of M-Elution Buffer directly to each well.  Wait 5 minutes, then centrifuge at ≥ 3,000 x g for 3 minutes 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 30 µl can be used if necessary.  The elution volume can be < 30 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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