4.2. Genotyping

Tail snips of 18–20-day old mice were soaked in 90 μL of 50 mM NaOH, heated for 10 min at 98 °C, and neutralized with 10 μL of 1M Tris (pH 8.0). Primers used for Stat3^flox mice were F1L (5′-aattggaacctgggaccaagtggccg-3′) and R2L (5′-agctggctcataggcaaaaacacctg-3′), and those used for Gdf9-iCre Tg mice were iCreF1 (5′-tggatgccacctctgatgaagtcag-3′) and iCreR1 (5′-tgattctcctcatcaccagggacac-3′). PCR was performed for 30 cycles at 98 °C for 10 s and 68 °C for 90 s using the EmeraldAmp PCR Master Mix (TaKaRa Bio Inc., Shiga, Japan). For oocyte genotyping, two GV oocytes, after removal of the zona pellucida by acidic Tyrode’s solution (T1788, Sigma-Aldrich, St. Louis, MO, USA), were introduced into a PCR tube containing 1.5 μL 50 mM NaOH and heated for 5 min at 95 °C After adding 6 μL of 30 mM Tris (pH 8.0), PCR was conducted for 35 cycles, as described above.