EpiTect Fast DNA Bisulfite Kit (50)

Protocol tips

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

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

Publication protocol

Methylation-specific PCR
Genomic DNA was extracted from Hep3B cells using the DNeasy tissue extraction kit (Qiagen) following the manufacturer’s instructions. Subsequently, 0.5–1.0 μg of DNA from each sample was used for bisulphite conversion using the EpiTect fast bisulfite conversion kit (Qiagen). The converted DNA was then purified with the same kit from Qiagen. DNA methylation status of the SPRY2 gene was examined by PCR employing two sets of primers that match the same sites with one specific for methylated (M) and the other for unmethylated (U) sequences. The sequences of primers are listed in S2 Table. Two rounds of PCR amplification were performed to detect the methylation status using FastStart PCR master kit (Roche Life sciences). The first round PCR amplification conditions used were one cycle of 95°C for 4 min followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 60 s. The resultant PCR product was diluted 20 times and used as template for the second round PCR amplification, which employed the nested forward primers (n) and the same reverse primers as in the first round PCR. The PCR conditions used were one cycle of 95°C for 4 min followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 60 s. The PCR products were loaded into agarose gels and the DNA methylation status was quantified by densitometry of the bands and normalized with beta-actin.



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Manufacturer protocol

Download the product protocol from Qiagen for EpiTect Fast DNA Bisulfite Kit (50) below.

Download manufacturer protocol

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