EZ DNA Methylation-Gold Kit

Protocol tips

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	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

Genomic DNA Preparation.
Live cultures of F. nucleatum obtained from DSMZ were propagated in modified peptone-yeast extract-glucose (PYG) medium (www.dsmz.de) in Anaerobe Atmosphere Generating Bags (Oxoid) for 48 h and then harvested by centrifugation. E. coli ER2566 was cultured in LB broth. Genomic DNA was isolated using the Genomic Mini DNA isolation kit (A&A Biotechnology). Genomic DNA of M. crocodyli, M. agalactiae PG2, M. penetrans HF-2, M. pulmonis UAB CTIP, M. mycoides ssp. capri, and M. infernus ME was obtained from D. Brown (University of Florida, Gainesville, FL), C. Citti (INRA ENVT, Toulouse, France), Y. Sasaki (National Institute of Infectious Diseases, Tokyo, Japan), P. Sirand-Pugnet (Université Bordeaux, Bordeaux, France), F. Thiaucourt (Centre International de Recherche en Agronomie pour le Développement, Montpellier, France), and W. Whitman (University of Georgia, Athens, GA), respectively.

DNA Methylation in Vitro.
Two micrograms of phage λ or E. coli genomic DNA was suspended in 49 μL of 10 mM Tris·HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, and 160 μM SAM; and methylated with 1 μL of M.MpeI (1 mg/mL), 1 μL of M.SssI (4 U/μL; NEB) for 4 h at 37 °C, or left unmethylated. Product was purified by chloroform extraction, precipitated with LiCl/ethanol, and suspended in water.

Bisulfite Conversion and Sample Workup.
Bisulfite conversion was carried out using the EZ Methylation Kit Gold (Zymo Research). For the analysis of methylation at randomly selected sites by Sanger sequencing, we used MethPrimer (33) designed primers (SI Appendix, Table S6) and DreamTaq DNA polymerase (Fermentas) for PCR amplification. For the analysis of methylation at the genome level by reversible terminator sequencing, we amplified bisulfite converted genomic DNA using the GenomePlex Whole Genome Amplification Kit (Sigma). The preparation of Illumina MiSeq libraries and the actual sequencing were carried out by Genomed as a commercial service.

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Manufacturer protocol

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

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