EZ DNA Methylation-Gold 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.	Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
Protocol tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

Quantitative methylation analyses by direct sequencing:

350 ng of genomic DNA was bisulfite converted using the EZ DNA Methylation-Gold kit (Zymo Research) according to the manufacturer’s instructions and eluted with 25 µL of the kit’s elution buffer. Two µL of the converted DNA was amplified with primers for Bisulfite Modified (BM) DNA, designed with MethPrimer software44 Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002; 18:1427 - 31; http://dx.doi.org/10.1093/bioinformatics/18.11.1427; PMID: 12424112
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
(primers sequences and PCR conditions are listed in Table S3). Five µL of the PCR reactions was treated with 2 µL of Exo-SAP-IT (GE Healthcare) to eliminate excess primers and nucleotides47 Jiang M, Zhang Y, Fei J, Chang X, Fan W, Qian X, et al. Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces. Lab Invest 2010; 90:282 - 90; http://dx.doi.org/10.1038/labinvest.2009.132; PMID: 20010852
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
(37°C, 15 min; 80°C, 15 min) and sequenced with the forward primer in an ABI 3130xl sequencer. Sequences were subject to visual quality control and areas with wide/overlapping peaks were excluded from subsequent analyses. A further quality control was performed with BiQ Analyzer software48 Bock C, Reither S, Mikeska T, Paulsen M, Walter J, Lengauer T. BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 2005; 21:4067 - 8; http://dx.doi.org/10.1093/bioinformatics/bti652; PMID: 16141249
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
to ensure a minimum of 80% identity match with the expected PCR product sequence and a minimum of 90% conversion rate of unmethylated cytosines to thymines. Cytosine methylation content was determined from the chromatograms applying the following formula:

% C Methylation = C peak height / (C peak height + T peak height) * 100.47

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Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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