The mixture was denatured at 95°C for 3 min then amplified by 20 cycles of denaturing at 95°C for 30 s, annealing at 62°C for 30 s and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min.
The reaction mixture was denatured at 95 °C for 3 min, followed by 26 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, with a final cycle of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 5 min [ 8].
The mixture was denatured at 95 °C for 3 min; and incubated on ice for 5 min; then, 2 μl was loaded onto a 6% acrylamide sequencing gel and the gel was run for 2 h in a 50-lane Applied Biosystems 373 A DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
The reaction mixture was denatured at 65°C for 10 minutes and cooled at 4°C for 5 minutes.
The PCR mixture was denatured for 5 min at 94°C and cycled 31 times (94°C for 30 sec, 55°C for 1.15 min and 72°C for 15 sec), followed by a 10 min extension at 72°C.
The ds cDNA mixture was denatured at 95°C for 10 min, and then incubated for 5 h at 60°C (for DRA and DQB) or 65°C (for DRB and DQA).
The mixture was denatured at 95°C for 5 min followed by rapid cooling on ice. 5 µl of 2.5 mM CoCl2, 5 µl of 10× terminal transferase (TdT) buffer (New England BioLabs), 5 µl of 50 µM dATP and 1.5 µl of TdT (20 U/µl, New England BioLabs) was then added and the samples were incubated at 42°C for 1 hr, and at 70°C for 10 min. The 3' ends of the polyA-tailed cDNA were then blocked with biotin-ddATP.
The mixture was denatured at 85 °C as before.
The probe mixture was denatured for 10 min at 65°C and immediately cooled on ice.
The mixture was denatured at 95°C for 4 min before being loaded on the gel.
Before being applied to the array, this mixture was denatured (>95°C, 5 minutes).
