EpiTect Fast LyseAll Bisulfite Kit (200)

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Protocol tips
Follow manufacturer's instructions

Publication protocol

Bisulfite Modification and Methylation Specific PCR (MSP). 1 × 105 cells were harvested and dissolved in 100 mL 5% PBS was used for further experiment. EpiTect Fast LyseAll Bisulfite Kit (Qiagen) was used to carry out bisulfite modification as per the manufacturer’s instructions. The modified DNA samples were finally stored at −20 °C until further use. The methylation status of the cell lines was determined using MSP. Primer sequences used for MSP in this study are shown in Supplementary Table 2. Polymerase chain reaction (PCR) was carried out in a volume of 50 μl high GC buffer with 100 ng or less template DNA with TaKaRa EpiTaq HS (TaKaRa). There were 35 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C. The amplified product was assayed using DNA gel electrophoresis and photographed using a Gel Doc chemiluminescence detector (BioRad). The qRT-PCR reaction was performed using SYBR Green core PCR reagents and bisulfite modified genomic DNA as a template and methylated GAPDH was used as the endogenous control. Primer sequences used for qRT-PCR for methylation study are provided in Supplementary Table 2.

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Manufacturer protocol

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