Aging Aging

330 Middleton, Curran, and WilliamsTable 3PCR Amplification ConditionsNo. ofLocus Hold Cycle cycles Hold HoldHLA-A 96°C/5 min 96°C/1 mingeneric 60°C/30 s 35 72°C/5 min 15°C/forever72°C/1 minHLA-B 96°C/5 min 96°C/30 sgeneric + 65°C/30 s 72°C/5 min 15°C/foreverHLA-B27 72°C/45 s 32TestingHLA-C 96°C/5 min 96°C/1 mingeneric 66°C/30 s 30 72°C/5 min 15°C/forever72°C/1 minHLA-DRB 96°C/5 min 96°C/1 mingeneric 55°C/1 min 30 72°C/5 min 15°C/forever72°C/1 minHLA-DR 3/11/6 96°C/5 min 96°C/1 min64°C/1 min 10 72°C/5 min 15°C/forever72°C/1 minthen96°C/1 min56°C/1 min 2072°C/1 min2. Carefully place membranes DNA face up onto two sheets thick (3MM) Whatmanpaper soaked in 0.4 M NaOH. Leave for 10 min. When placing membranes ontoWhatman paper, take care to ensure that membrane is not dragged over denaturationpad, all of the membrane soaks up the 0.4 M NaOH, and there are no airbubbles beneath the membrane.3. Transfer each membrane onto Whatman paper (3MM) soaked in 10× SSPE. Leavefor 5 min.4. Gently wash in 2× SSPE and allow to air-dry for at least 25 min.5. Wrap membranes in Saran Wrap and place (DNA face down) on UV transilluminatorfor 4 min. Ensure that all the UV lights are fully on during the procedure;do not switch transilluminator off between each step. Place a glass plate on top ofmembranes to hold them flat during this procedure. Store membranes wrapped intin foil at +4°C if not using immediately.