Methylation gold kit

Genomic DNA was extracted from the lungs of the three deceased transgenic cloned kids and the normally produced kids using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China), and this was followed by sodium bisulfite treatment using the Methylation-Gold™ Kit. Subsequently, the modified DNA sample was immediately used in bisulfite sequencing PCR (BSP) or stored at -80°C.
Specific primers, which were designed using MethPrimer, were used for the BSP amplification of IGF2R. Details of BSP-amplified nucleotide sequences of the IGF2R ICR are shown in Figure 1. PCR was performed with a DNA engine (Bio-Rad, USA) using the following program: 5 min at 95°C, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 51°C, and extension for 30 s at 72°C. A final extension was run at 72°C for 5 min. The PCR products were gel-purified using the AxyPrep Purification Kit (Axygen, USA). The purified fragments were subcloned into pMD18-T vectors (TaKaRa, Japan). The clones confirmed by PCR were sequenced. We sequenced ten clones from each independent set of amplification and cloning. BSP results and C-T conversion rate were analyzed by the BIQAnalyzer software (http://biq-analyzer.bioinf.mpi-inf.mpg.de/). To ensure data quality, we chose sequences for which the C-T conversion rate was greater than 95% (Figure 1).