About EZ-96 DNA Methylation-Gold™ MagPrep
The EZ-96 DNA Methylation-Gold™ MagPrep integrates DNA denaturation and bisulfite conversion processes into one-step coupled to a magnetic bead based clean-up for high-throughput methylation analysis. This is accomplished using temperature denaturation to replace chemical denaturation with sodium hydroxide in the previous protocols. Also, the kit has been streamlined for high yield recovery of DNA following DNA bisulfite conversion. 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 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 130 µl of CT Conversion Reagent to 20 µl* of a DNA sample in a Conversion Plate. 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:
1. 98 °C for 8 minutes
2. 54 °C for 60 minutes
3. 4 °C storage for up to 20 hours
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°C-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.