About EZ-96 DNA Methylation-Lightning™ MagPrep
The EZ-96 DNA Methylation-Lightning™ MagPrep Kit features rapid and reliable bisulfite treatment and conversion of DNA coupled to a magnetic bead based clean-up for high-throughput methylation analysis. Key to the fast workflow is the ready-to-use Lightning Conversion Reagent. No preparation is necessary, simply add this unique reagent to a DNA sample, wait about an hour, and let the reaction proceed to completion. DNA denaturation and bisulfite conversion processes are combined with added heat to facilitate rapid denaturation. Desulphonation and clean-up of the converted DNA is performed while bound to the MagBinding Beads. High yield, converted 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|
|Conversion Efficiency||>99.5% of non-methylated cytosine residues are converted to uracil; >99.5% protection of methylated cytosines.|
|Optimal DNA Input||Samples containing between 100 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 Lightning 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 L-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.
“It combines the speed of the lightning kit with the use of magnetic beads. This allows the full automation of bisulfite treatment. This is what we were looking for.”
Marco M. (UCLA)