About EZ-96 DNA Methylation-Direct™ Kit
The EZ-96 DNA Methylation-Direct™ Kit is a further refinement of our popular EZ DNA Methylation™ and EZ DNA Methylation-Gold™ kits. These products feature reliable and complete DNA bisulfite conversion directly from blood, tissue, and cells without the prerequisite for DNA purification. The increased sensitivity of these kits make it possible to amplify bisulfite-converted DNA from as few as 10 cells or 50 pg DNA. Like the EZ DNA Methylation-Gold™ kits, DNA denaturation and bisulfite conversion processes are combined into a single step. Recovered bisulfite-converted DNA is ideal for PCR amplification for downstream analyses including restriction endonuclease digestion, sequencing, microarrays, etc.
|Learn More About Bisulfite Conversion||Click to see which kit is right for you|
|DNA Recovery||> 80%|
|Processing Time||4 hrs|
|Equipment||Centrifuge with microplate carrier|
|Binding Capacity||5 µg/well|
|Elution Volume||Shallow-well: ≥ 30 µl, Deep-well: ≥ 15 µl|
|Conversion Efficiency||> 99.5%|
|Input||DNA, cells, blood, tissue, FFPE|
EZ DNA Methylation-Direct™ Kit bisulfite chemistry significantly improves C to U conversion kinetics. DNA was converted using either EZ DNA Methylation-Direct™ or conventional bisulfite chemistries. Recovered DNA was amplified by PCR, then cloned. Sequences from individual clones were analyzed and quantitated. These data show that EZ DNA Methylation-Direct™ bisulfite chemistry improves the rate and extent (< 99.8%) of C to U conversion of DNA as compared to conventional bisulfite chemistry.
Either blood, tissue, cells, or purified DNA can be used as the starting material for the EZ-96 DNA Methylation-Direct™ Kit. If purified DNA is used, then proceed directly to Section II.
Section I: Sample Digestion with Proteinase K
1. Set up Proteinase K digestions for your specific sample type (please see detailed protocol for more information). Digestions should be performed in the provided Conversion Plate.
2. Seal the Conversion Plate with Cover Film and incubate the samples for 20 minutes at 50°C.
Section II. Bisulfite Conversion of DNA
1. Add 130 μl of CT Conversion Reagent solution to each 20 μl sample in the Conversion Plate from Section I.
2. Seal the Conversion Plate with Cover Film. Place the plate in a thermal cycler and perform the following steps:
1. 98°C for 8 minutes
2. 64°C for 3.5 hours
3. 4°C storage for up to 20 hours
3. Add 600 μl of M-Binding Buffer to the wells of the Binding Plate on a provided Collection Plate.
4. Load the samples (from Step 2) into the wells of the Binding Plate containing the M-Binding Buffer. Mix by pipetting up and down.
5. Centrifuge at ≥ 3,000 x g (5,000 x g max.) for 5 minutes. Discard the flow-through and reuse the Collection Plate for the following wash steps.
6. Add 400 μl of M-Wash Buffer to each well of the plate. Centrifuge at ≥ 3,000 x g for 5 minutes.
7. Add 200 μl of M-Desulphonation Buffer to each well and let the plate stand at room temperature (20-30°C) for 15-20 minutes, then centrifuge at ≥ 3,000 x g for 5 minutes. Discard the flow-through.
8. Add 400 μl of M-Wash Buffer to each well of the plate. Centrifuge at ≥ 3,000 x g for 5 minutes. Add another 400 μl of M-Wash Buffer and centrifuge for an additional 5 minutes.
9. Place the Binding Plate onto a provided Elution Plate. Add 15 μl of M-Elution Buffer directly to each well. Wait 5 minutes, then centrifuge for 3 minutes at ≥ 3,000 x g 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 ≤ -70°C. We recommend using 1-4 μl of eluted DNA for each PCR, however, 15 μl can be used if necessary. The elution volume can be > 15 μl depending on the requirements of your experiments, but small elution volumes will yield higher concentrations of DNA.