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EZ-96 DNA Methylation™ MagPrep

Proven procedure for complete bisulfite conversion of DNA.
High throughput (96-well), automated desulphonation and recovery of bisulfite-treated DNA.
Recovered DNA is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.

Product Size Catalog # Price Qty
EZ-96 DNA Methylation™ MagPrep 4x96 Rxns D5040
EZ-96 DNA Methylation™ MagPrep 8x96 Rxns D5041

About EZ-96 DNA Methylation™ MagPrep

The EZ-96 DNA Methylation™ MagPrep features a high-throughput (96-well), simplified procedure that streamlines bisulfite conversion of DNA. The kit is based on the three-step reaction that takes place between cytosine and sodium bisulfite where cytosine is converted into uracil. Desulphonation and clean-up of the converted DNA is performed while bound to the MagBinding Beads. The kit is designed to reduce template degradation, minimize DNA loss during treatment and clean-up, while ensuring complete conversion of the DNA. Purified, converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion, sequencing, microarrays, etc.

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

Equipment Magnetic Stand, Heating element for 96-well plate.
Conversion Efficiency >99% of non-methylated cytosine residues are converted to uracil; >99% protection of methylated cytosines.
Optimal DNA Input Samples containing between 500 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 5 µl of M-Dilution Buffer to each DNA sample in a Conversion Plate and adjust the total volume to 50 µl with water.  Mix each sample by pipetting up and down.

Step 2

Incubate the Conversion Plate containing the samples at 37°C for 15 minutes.

Step 3

After the above incubation, add 100 µl of the prepared CT Conversion Reagent to each sample and mix.

Step 4

Seal the plate with the provided film.  Incubate the Conversion Plate in the dark at 50°C for 12-16 hours (e.g., using a thermal cycler).

The CT Conversion reagent is light sensitive, so try to minimize the reaction’s exposure to light whenever possible.

Step 5

Incubate the sample at 0-4°C (e.g., on ice or using a thermal cycler) for 10 minutes.

Step 6

Pre-heat a plate heating element to 55°C.

Step 7

Add 600 µl of M-Binding Buffer and 10 µl of MagBinding Beads to each well of a Collection Plate.

Step 8

Transfer the samples from the Conversion Plate into the Collection Plate containing the M-Binding Buffer and MagBinding Beads.  Mix by pipetting up and down 3-6 times and, if available, vortexing at 1,300-1,500 rpm for 30 seconds (e.g. Tecan - Te-Shake™).

Step 9

Let plate stand at room temperature for 5 minutes, then transfer plate to a magnetic stand for an additional 5 minutes or until beads pellet and supernatant is cleared.  With the plate on the magnetic stand remove the supernatant and discard.

Step 10

Remove the Collection Plate from the magnetic stand for this and each subsequent buffer addition.  Add 400 µl of M-Wash Buffer to the beads.  Re-suspend the beads by pipetting up and down or vortexing the plate at 1,300-1,500 rpm for 30 seconds.  Replace the plate on the magnetic stand for 3 minutes or until beads pellet.  Remove and discard supernatant.

Step 11

Add 200 µl of M-Desulphonation Buffer to the beads.  Re-suspend the beads by pipetting up and down or vortexing for 30 seconds.  Let plate stand at room temperature (20-30°C) for 15-20 minutes.  After the incubation, replace the plate on the magnetic stand for 3 minutes or until beads pellet.  Remove and discard supernatant.

Step 12

Add 400 µl of M-Wash Buffer to the beads.  Re-suspend the beads by pipetting up and down or vortexing for 30 seconds. Replace the plate on the magnetic stand for 3 minutes or until beads pellet.  Remove and discard supernatant.  Repeat this wash step.

Step 13

Transfer the plate to a heating element at 55°C for 20-30 minutes to dry the beads and remove residual M-Wash Buffer.

Step 14

Add 25 µl of M-Elution Buffer directly to the dried beads and pipette or vortex for 30 seconds to re-suspend.  Heat the elution at 55°C for 4 minutes then transfer the plate to the magnetic stand for 1 minute or until beads pellet.  Remove the supernatant and transfer to a clean Elution Plate.

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 25 µl can be used if necessary.  The elution volume can be < 25 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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