Pre-denature 95 5 min 95 5 min ℃ ℃ Denature 95 30 s 95 30 s ℃ ℃ Annealing 57 30 s 60 30 s ℃ ℃ Extension 72 1 min 72 1 min ℃ ℃ Cycles 30 35 Extension 72 7 min 72 7 min ℃ ℃ Storage 4 4 ℃ ℃ S3. Restriction endonuclease digestion conditions rs6294 rs6298 Reaction Volume (μL) 20 20 Restriction Enzyme (μL) RsaI, 0.5 BccI, 0.5
Pre-denature 95 ℃ 5 min 95 ℃ 5 min Denature 95 ℃ 30 s 95 ℃ 30 s Annealing 57 ℃ 30 s 60 ℃ 30 s Extension 72 ℃ 1 min 72 ℃ 1 min Cycles 30 35 Extension 72 ℃ 7 min 72 ℃ 7 min Storage 4 ℃ 4 ℃ S3. Restriction endonuclease digestion conditions rs6294 rs6298 Reaction Volume (μL) 20 20 Restriction Enzyme (μL) RsaI, 0.5 BccI, 0.5