EZ DNA Methylation 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

Sorted PGCs were bisulfite-treated with the EZ DNA Methylation-Direct Kit (Zymo Research, Irvine, CA). Sequences of the PCR primers are listed in Supplemental Table S1 (all the supplemental data are available online at www.biolreprod.org). PCR amplification of IAP (GenBank accession no. M17551) and Line-1 (GenBank accession no. D84391) was carried out with Ex Taq (Takara Bio, Shiga, Japan) under the following conditions: 5 min at 95°C followed by 30 cycles of PCR consisting of 1 min at 95°C, 2 min at 56°C, and 1 min at 72°C. Fully or seminested PCR was performed to amplify the H19 (GenBank accession no. U19619) and Rasgrf1 (GenBank accession no. AF021791) differentially methylated regions (DMRs). The first and second rounds of PCR were carried out with AccuPrime Taq DNA polymerase (Invitrogen) and Ex Taq (Takara Bio), respectively. The PCR conditions were as follows: the first round of 2 min at 94°C followed by 30 cycles consisting of 30 s at 94°C, 30 s at 50°C, and 1 min at 68°C, and the second round of 2 min at 95°C followed by 15 cycles consisting of 30 s at 95°C, 30 s at 50°C, and 1 min at 72°C. The PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), cloned into the pGEM-T Easy Vector (Promega, Madison, WI), and then sequenced using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The number of PGCs and the embryos in each experiment are summarized in Supplemental Table S2. Primer information is shown in Supplemental Table S3.

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Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation kit below.

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