Aging Aging

Immunogenetics and Life-Span 329Table 2 (cont.)Locus Stock conc Master mixHLA-C genericddH 2 O 8300µLCresol red 10 mg/mL 100 µLNH 4 buffer 10× 1000 µLMgCl 2 50 mM 300 µLdNTPs 20 mM each 100 µLEach primer ×2 25µM 80 µLTaq 5 U/µL 40µLHLA-DRB genericddH 2 O 8280µLCresol red 10 mg/mL 100 µLNH 4 buffer 10× 1000 µLMgCl 2 50 mM 300 µLdNTPs 20 mM each 100 µLEach primer (×2) 25 µM 100 µLTaq 50 U/µL 20µLHLA-DR3/11/6ddH 2 O 8300µLCresol red 10 mg/mL 100 µLNH 4 buffer 10× 1000 µLMgCl 2 50 mM 300 µLdNTPs 20 mM each 100 µLEach primer ×2 25µM 80 µLTaq 5 U/µL 40µLthe required number of membranes proves difficult an alternative method is todehybridize used membranes as follows.1. Dehybridize a maximum of three membranes in 300 mL of each solution with shaking.2. Rinse membranes in ddH 2 O for 5 min at room temperature.3. Wash membranes in 0.4 M NaOH/0.1% SDS at 45°C for 30 min.4. Wash membranes in 2× SSC for 30 min at room temperature.5. Check dehybridization is complete by exposing membranes overnight to X-rayfilm and developing in usual manner.6. Store membrane flat at +4°C in a sealed plastic bag if not using immediately.3.4. Denaturation and Fixing of Blots1. After PCR product has been dispensed onto the membranes, allow to air-dry forat least 20 min.