Methylation Sensitive PCR

1. The percentage methylation calculated for each embryo is obtained by comparing the band intensity of an undigested DNA PCR product (representing 100% methylation) with the HhaI-digested PCR product.

2. Perform quadruplicate PCRs in a 25-|L volume containing 12.5 |L of 2X Thermostart mastermix, 250 nM each primer (2.5 ||L), and 2 |L of either HhaI-digested or -undigested DNA.

3. Perform PCR in a thermal cycler with a heated lid (to avoid using mineral oil) at 1 cycle of 15 min at 94°C, followed by repeated cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 45 s. For OVSAT1 primers (see Note 2) the optimal cycle number in our hands was 19 for blastocysts, 21 for morulae, 22 for 8- to 16-cell embryos, and 24 for oocytes. In all cases, methylation values were normalized against the relevant standard curve (see Subheading 3.5.).

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