About EZ-96 DNA Methylation-Direct™ MagPrep
The EZ-96 DNA Methylation-Direct™ MagPrep features simple and reliable DNA bisulfite conversion directly from blood, tissue, and cells without the prerequisite for DNA purification. The increased sensitivity of this kit makes it possible to amplify bisulfite converted DNA from as few as 10 cells or 50 pg DNA. These innovations have been coupled to a magnetic bead based clean-up for high-throughput methylation analysis. Desulphonation and clean-up of the converted DNA is performed while bound to the MagBinding Beads. The kits have been designed to minimize template degradation, loss of DNA during treatment and clean-up, and to provide complete conversion of unmethylated cytosines. Recovered 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|
|Equipment||Magnetic Stand, Heating element for 96-well plate.|
|Conversion Efficiency||>99.5% of non-methylated cytosine residues are converted to uracil; >99.5% protection of methylated cytosines.|
Either blood, tissue, cells, or purified DNA can be used as the starting material for the EZ-96 DNA Methylation-Direct™ MagPrep 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. Pre-heat a plate heating element to 55°C.
4. Add 600 μl of M-Binding Buffer and 10 μl of MagBinding Beads to each well of a Collection Plate.
5. Transfer the samples from the Conversion Plate (from Step 2) 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™).
6. 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.
7. 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.
8. 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.
9. 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.
10. Transfer the plate to a heating element at 55°C for 20-30 minutes to dry the beads and remove residual M-Wash Buffer.
11. 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.
Various automation scripts are available and can be obtained free of charge by contacting Zymo Research at firstname.lastname@example.org. Include “Automation Scripts” in the subject line and provide kit catalog number and the automation platform desired in the email.