About EZ-96 DNA Methylation-Gold™ Kit
The EZ-96 DNA Methylation-Gold™ Kit is a refinement of our popular EZ DNA Methylation™ kits. These products consolidate DNA denaturation and bisulfite conversion processes into one step, leading to a much faster bisulfite conversion. This is accomplished using temperature denaturation to complement chemical denaturation in the previous protocol. Also, this kit have been streamlined for high yield recovery of DNA following bisulfite treatment. Recovered bisulfite-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|
|DNA Recovery||> 75%|
|Processing Time||3 hrs|
|Equipment||Centrifuge with microplate carrier|
|Binding Capacity||5 µg/well|
|Elution Volume||Shallow-well: ≥ 30 µl, Deep-well: ≥ 15 µl|
|Conversion Efficiency||> 99%|
|Input||Plasmid, genomic, and endonuclease digested DNAs.|
DNA sequencing results after bisulfite treatment. DNA with methylated CmpG (at nucleotide position #5) was processed using the EZ DNA Methylation-Gold™ Kit. The recovered DNA was amplified by PCR and then sequenced directly. The methylated cytosine at position #5 remained intact while the unmethylated cytosines (i.e., positions #7, 9, 11, 14 and 15) were completely converted into uracil following bisulfite treatment and detected as thymine following PCR.
Add 130 µl of the CT Conversion Reagent to 20 µl of each DNA sample in a Conversion Plate. If the volume of the DNA sample is less than 20 µl, make up the difference with water. Mix the samples by pipetting up and down.
Seal the plate with the provided film. Transfer the Conversion Plate to a thermal cycler and perform the following steps*:
2. 64°C for 2.5 hours
3. 4°C storage up to 20 hours
*For some samples, alternative parameters may yield improved results (see Appendix). If you have been using this kit with good results using different reaction conditions than described above, you can continue using those same conditions.
Add 400 µl of M-Binding Buffer to the wells of a Silicon-A™ Binding Plate mounted on a Collection Plate.
Transfer the samples from the Conversion Plate (Step 2) to the wells of the Silicon-A™ Binding Plate. Mix by pipetting up and down.
Centrifuge at ≥ 3,000 x g (5,000 x g max.) for 5 minutes. Discard the flow-through.
Add 400 µl of M-Wash Buffer to each well of the plate. Centrifuge at ≥ 3,000 x g for 5 minutes.
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.
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.
Place the Silicon-A™ Binding Plate onto an Elution Plate. Add 30 µl of M-Elution Buffer directly to each well. After 5 minutes, 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 more concentrated DNA.
The authors used the EZ-96 DNA Methylation-Gold™ Kit to bisulfite convert lymphocyte DNA and subsequently analyzed the bisulfite-treated DNA with the Illumina Infinium HumanMethylation450 BeadChip Array (450k array). In this study, the authors evaluated the quality and characteristics of the probes used in the 450k array, especially with respect to detection of sex-specific differences in DNA methylation levels.