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.|
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.
Incubate the Conversion Plate containing the samples at 37°C for 15 minutes.
After the above incubation, add 100 µl of the prepared CT Conversion Reagent to each sample and mix.
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.
Incubate the sample at 0-4°C (e.g., on ice or using a thermal cycler) for 10 minutes.
Pre-heat a plate heating element to 55°C.
Add 600 µl of M-Binding Buffer and 10 µl of MagBinding Beads to each well of a Collection Plate.
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™).
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.
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.
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.
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.
Transfer the plate to a heating element at 55°C for 20-30 minutes to dry the beads and remove residual M-Wash Buffer.
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.