CpGenome Universal DNA Modification 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.

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

Publication protocol

The genomic DNA of the cells was extracted using a DNA extraction kit according to the manufacturer’s instructions. Following identification and quantification by UV spectrophotometry, 1 μg DNA was collected to perform the bisulfate conversion with a CpGenome™ DNA Modification kit (Millipore), according to the manufacturer’s instructions. PCR was used to amplify 287 bp from the binding region of the PTEN promoter region and the transcription factor Myc in the bisulfate-converted DNA. The amplification primer sequences were: 5′-TATTTATAAGGTGGAAGTTTTGAGG-3′ and 5′-ATAAAAAATAAACTCAACCCCACTC-3′. The PCR amplification conditions were 94°C for 3 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec; and a final extension at 72°C for 7 min. The PCR products were cloned into a T-vector and transformed into Escherichia coli (E. coli) cells (DH5α). Subsequently, the E. coli were inoculated in Ampicillin+ (100 μg/ml) LB agar plates, incubated at 37°C for 12–16 h and then five independent clones were sequenced for the amplified fragment. The demethylation rate of the CpG pairs in the MG-63 cells treated with or without different concentrations of 5-Zac was calculated from the sequencing results.

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Manufacturer protocol

Download the product protocol from Millipore for CpGenome Universal DNA Modification Kit below.

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