EZ DNA Methylation-Direct 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.

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

Publication protocol

Methylation-sensitive genotyping
5hmC- 5 μg of heterozygous placenta DNA was subject to DNA Glucosylation using the Epimark kit (New England Biolabs) and the DNA subject to digestion with 100 units of Msp1 for a minimum of 8 hours at 37°C. The DNA was subject to proteinase K digestion prior to PCR.

5mC- Approximately 1 μg of heterozygous placenta DNA was digested with 10 units of HpaII restriction endonuclease for 6 hours at 37°C. The digested DNA was subject to ethanol precipitation and resuspended in a final volume of 20 μl TE. Approximately 50 ng of digested DNA was used in each amplification reaction using Bioline Taq polymerase for 35–40 cycles (for primer sequences see S6 Table). The resulting amplicons were sequenced and the sequences traces compared to those obtained for the corresponding undigested DNA template.

Bisulphite PCR of placenta-derived DNA
For standard bisulphite conversion approximately 1 μg DNA was subjected to sodium bisulphite treatment and purified using the EZ DNA methylation-Gold kit (ZYMO, Orange, CA). Approximately 2 ul of bisulphite converted DNA was used in each amplification reaction using Immolase Taq polymerase (Bioline) at 45 cycles and the resulting PCR product cloned into pGEM-T easy vector (Promega) for subsequent subcloning and sequencing (for primer sequence see S6 Table).



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Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation-Direct Kit below.

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