Published in
BioTechniques Protocol Guide
2008
(p.27)
Overview
The EZ DNA Methylation-Direct™ Kit is a further refinement of our popular EZ DNA Methylation™ and EZ DNA Methylation-Gold™ Kits. The EZ DNA Methylation-Direct™ Kit 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. Specifications
Starting Materials:
Cells: Compatible with cells from solid tissue, tissue culture, whole blood, buffy coat, biopsies, LCM (Laser-Capture Micro-Dissection) and FFPE samples, etc. The number of cells per standard treatment can range from 10–105 cells. For optimal results, the cell number should be from 1×103– 8×104 cells.
Purified DNA: Samples containing 50 pg - 2 µg of DNA. For optimal results, the amount of input DNA should be from 200 to 500 ng.
Conversion Efficiency: >99.5% of non-methylated C residues are converted to U; >99.5% protection of methylated cytosines.
DNA Recovery: >80%
Sensitivity of Detection (Lower Limit): 10 cells for successful PCR amplification.
Protocol (Spin Column Format)
Sample Digestion with Proteinase K
Set up Digestion:
Incubate at 50°C for 20 minutes.
Mix by flicking or vortexing the tube. Spin for 5 minutes at 10,000 × g.
Bisulfite Conversion
Add 130 µl of the prepared CT Conversion Reagent to 20 µl of supernatant from Section I. Mix.
Perform the following temperature steps: 98°C for 8 minutes, 64°C for 3.5 hours, then hold at 4°C.
Add 600 µl of M-Binding Buffer to a Zymo-Spin™ IC Column and then add the sample. Close the cap and mix by inversion.
Centrifuge at full speed (>10,000 × g) for 30 seconds. Discard the flow-through.
Add 100 µl of M-Wash Buffer to the column and then centrifuge 30 seconds.
Add 200 µl of M-Desulphonation Buffer to the column and wait for 20 minutes.
Spin at full speed for 30 seconds.
Add 200 µl of M-Wash Buffer to the column, spin 30 seconds. Repeat this wash step.
Add 10 µl of M-Elution Buffer directly to the column matrix. Place into a 1.5 ml tube. Spin briefly to elute the DNA.
The DNA is ready for immediate analysis or can be stored at ≤ − 20°C for later use. Recovered DNA is ideal for PCR amplification for downstream analyses including restriction endonuclease digestion, sequencing, microarrays, etc.
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