Aging Aging

324 Middleton, Curran, and WilliamsIn this laboratory we do not use tetramethylammonium chloride (TMAC)owing to its toxic properties and the fact that in our experience it does notnecessarily mean the use of one wash temperature. It would appear that to followthis practice a laboratory would require a large number of water baths. Inthis laboratory one individual normally performs 12 hybridizations at the sametime. Thus if a laboratory is defining alleles at three loci (HLA-A, -B, -DR),probes can be selected for use at the same time according to their wash temperature.This eliminates the requirement for a large number of water baths.In the methods described each probe is hybridized to two different membranesin the same hybridization bottle and the reagents are prepared accordingly.The SSOP method is thus very suitable for typing large numbers ofsamples — we test at the same time 192 samples (96 on each membrane) whichincludes controls. However, the volume of reagents can be scaled down and ifa laboratory is not performing tests on large numbers of samples, only one lotof membranes need be hybridized.2. Materials1. Buffer 1 (4×): 0.4 M Maleic acid, 0.6 M NaCl, pH 7.5. Add 300 mL of 4 M NaCland 400 mL of 2 M maleic acid followed by 200 mL of 4 M NaOH to approx 800 mLof double-distilled H 2 O (ddH 2 O). Add 27 g of NaOH pellets. (Note: A whiteprecipitate forms when all reagents are added — this will disappear as the pHapproaches 7.0.) Cool to room temperature and adjust pH to 7.5 by adding 4 MNaOH by drops. Adjust volume to 2 L with ddH 2 O and sterilize by autoclaving.2. Buffer 2: 2% Blocking reagent in buffer 1. Combine 768 mL of 5% blockingreagent (in buffer 1), 288 mL of 4× buffer 1, and 864 mL of ddH 2 O. Leave 5%blocking reagent at room temperature for 10 min before use.3. Buffer 3: 0.1 M Tris-HC1, 0.1 M NaCl, 0.05 M MgCl 2 , pH 9.5. Add approx 1400 mLof ddH 2 O to 200 mL of 1 M Tris-HCl, pH 9.5, and 50 mL of 4 M NaCl. Add100 mL of filter sterilized 1 M MgCl 2 and mix. Adjust pH to 9.5 and make up to2 L with ddH 2 O. Do not autoclave as precipitates tend to form. Store at roomtemperature for up to 1 wk.4. Buffer hybridization: 192 mL of 2% blocking reagent, 144 mL of 6× salinesodium phosphate EDTA (SSPE), 48 mL of 5× Denhardts, 48 mL of 0.1%N-laurylsarcosine, 0.96 mL of 0.02% sodium dodecyl sulfate (SDS) and make upto 480 mL with ddH 2 O.5. Buffer washing: 0.3% Tween-20 in buffer 1. Add 14.4 mL of Tween-20 to 1200 mLof 4× buffer 1 and make up to 4800 mL by adding ddH 2 O.6. Blocking reagent: 5% in buffer 1 (Boehringer, Lewes, England, cat. no. 1096176).Prepare 2 L of 1× buffer 1 by combining 500 mL of 4× buffer 1 with 1500 mL ofddH 2 O. Add 100 g of blocking reagent in parts, with vigorous mixing using amagnetic stirrer, to approx 1600 mL of 1× buffer 1. As blocking reagent is suppliedin 50 g tubs, there is no need to weigh out. Heat to 65°C until blocking