EMD Millipore™ Chemicon™ CpGenome™ Universal DNA Modification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Publication protocol

2.4. DNA Extraction
Genomic DNA was extracted from cell lines and tissue samples using a commercial DNA extraction kit (QIAmp Tissue Kit; Qiagen, Hilden, Germany). DNA was isolated according to the manufacturer's protocol.

2.5. Bisulfite Modification and Methylation-Specific PCR (MS-PCR)
Genomic DNA isolated from cells and tissue was subjected to bisulfite methylation analysis. We treated DNA with bisulfite using an EZ DNA methylation kit (Zymo Research, Orange, CA) according to the protocol described in the user manual. Briefly, one µg of genomic DNA was denatured by incubation with 0.2 M NaOH. Aliquots of 10 mM hydroquinone and 3 M sodium bisulfite (pH 5.0) were added and the solution was incubated at 50°C for 16 hr. Treated DNA was purified on a Zymo-Spin I column, desulfonated with 0.3 M NaOH, repurified on a Zymo-Spin I column, and resuspended in 20 μL elution buffer. MS-PCR [35] was carried out in a volume of 25 μL containing 1 μL of the sodium-bisulfite-treated DNA with Gold Taq DNA polymerase (PE Applied Biosystems, Foster City, CA) as follows. After heating at 92°C for 10 min, PCR was performed in a thermal cycler (GeneAmp 2400, PE Applied Biosystems) for 35 cycles, each of which consisted of denaturation at 92°C for 30 sec, annealing at 61°C for 30 sec, and extension at 72°C for 30 sec, followed by a final 10 min extension at 72°C. The PCR products were analyzed by electrophoresis on a 3% agarose gel. The experiments were repeated three times to ensure reproducibility.

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Manufacturer protocol

Download the product protocol from Fisher Scientific for EMD Millipore™ Chemicon™ CpGenome™ Universal DNA Modification Kit below.

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