EZ DNA Methylation-Gold Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

DNA Methylation Analysis.
Genomic DNA from tissue samples or cells was isolated using the DNeasy Blood and Tissue Kit (Qiagen). DNA was considered qualified with a ratio of absorbance at 260 nm and 280 nm (A260/A280) between 1.7 and 1.9. Samples were stored in elution buffer at −80°C before use.

Sodium bisulfite modification of DNA samples was conducted using an EZ DNA Methylation-Gold Kit (Zymo, Orange, CA). The location of all CpG sequences examined in this study is shown in Fig. 1. PCR amplification was done with rTaq polymerase (Takara) in a 50-μl reaction mixture with 2 μl modified DNA as the template. PCR amplification started with 10 cycles of touchdown PCR of 30 seconds at 94°C, 30 seconds at 60°C to 55°C (−0.5°C per cycle), and then 45 seconds at 72°C and 30 cycles of 30 seconds at 94°C, 30 seconds at 55°C, and 45 seconds at 72°C, followed by an extension of 10 minutes at 72°C. Amplicons were then purified and subcloned into pMD18-T vector (Takara) according to the manufacturer’s instructions. Ligation products were transformed into Escherichia coli DH5α cells and 10–15 random colonies with recombinant plasmids were subjected to Sanger sequencing (Sangon, Shanghai, China).
Methylation-specific PCR was also conducted using the modified DNA as a template. The following touchdown PCR program was applied: 10 cycles of touchdown PCR of 30 seconds at 94°C, 30 seconds at 58°C to 53°C (−0.5°C per cycle), 20 seconds at 72°C and 25 cycles of 30 seconds at 94°C, 30 seconds at 53°C, and 20 seconds at 72°C, followed by a final extension of 10 minutes at 72°C. Ten-microliter PCR products were shown on 2% agarose gel containing ethidium bromide.

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Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - HepG2 Constitutive Androstane Receptor (CAR) using EZ DNA Methylation-Gold Kit from Zymo Research.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - HepG2 Constitutive Androstane Receptor (CAR) using EZ DNA Methylation-Gold Kit from Zymo Research. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.