Aging Aging

Immunogenetics and Life-Span 325reagent is dissolved. Allow to cool to room temperature and make up to 2 L withbuffer 1. Sterlize by autoclaving and store at 4°C.7. Cresol red: 10 mg/mL, sodium salt (Sigma, St. Louis, MO, USA, cat. no. C9877).Add 200 mg to some ddH 2 O taken from measured 20 mL dH 2 O in a sterile bottle.Resuspend in remaining volume. Filter sterilize and dispense into 1-mL aliquotsand freeze at –20°C.8. CSPD (Boehringer, cat. no. 1655884). Vortex-mix and centrifuge CSPD in amicrocentrifuge for 1 min before use. Dilute CSPD stock solution (25 mM,.6 mg/mL) 1:100 in buffer 3.9. Denhardts (50×): 1% Polyvinyl pyrrolidone (PVP), 1% Ficoll, 1% bovine serumalbumin (BSA). Prepare 200 mL of 2% PVP and 2% Ficoll by adding 4 g of eachto 180 mL ddH 2 O. (Prepare this solution in a fume cupboard. PVP is harmful ifinhaled.) Dissolve with gentle mixing and make up to 200 mL with ddH 2 O. Sterilizeby autoclaving and cool to room temperature. Add 4 g of BSA to 200 mL ofthe above solution slowly with gentle mixing. When the BSA has dissolved makeup to 400 mL with ddH 2 O, mixing well. Filter solution through 0.45 µm filter andaliquot. Do not autoclave. Store at –20°C. Leave to thaw at +4°C the eveningbefore it is to be used.10. Anti-DIG–ALP conjugate (Boehringer, cat. no. 1093274). Just prior to useremove anti-DIG–ALP stock conjugate (0.75 U/µL) from the refrigerator, vortex-mixfor 15 s and centrifuge for 1 min in a microcentrifuge. Make a 1:10,000dilution of the conjugate in buffer 2 (i.e., 192 µL of anti-DIG–ALP conjugate in1920 mL of buffer 2).11. 0.5 M EDTA, pH 8.0. Add 186.1 g of EDTA Na 2 2H 2 O in parts to 800 mL ofddH 2 O. Adjust the pH to 8.0 using 4 M NaOH. Make up to 1 L with ddH 2 O andsterilize by autoclaving.12. Ethidium bromide (10 mg/mL, Sigma, cat. no. E-1510).13. Gel loading buffer (GLB): Add 8 g of sucrose (slowly) to 10 mL of ddH 2 O andmix by inversion until dissolved. Then add 1 mL of 1 M Tris, pH 7.6; 2 mL of 0.5 MEDTA; 1 mL of 10% SDS; 0.02 g of cresol red. Make up to 20 mL with ddH 2 O.Do not autoclave.14. ddH 2 O. Double-distilled H 2 O or equivalent. Note that ddH 2 O used to set up PCRis of ultra-high-purity quality.15. MgCl 2 . Supplied with Taq enzyme.16. dNTPs (Pharmacia Biotech, St. Albans, England, cat. no. 27-2094).17. NH 4 buffer. Supplied with Taq enzyme.18. N-Laurolysarcosine (1%). Barrier face mask should be worn when weighingN-laurylsarcosine. Dissolve 10 g of N-lauryl sarcosine in approx 800 mL ofddH 2 O. Adjust volume to 1 L with ddH 2 O and autoclave.19. Nylon membrane (Boehringer, cat. no. 1417 240).20. PCR plates, 96-well (Advanced Biotechnologies, Epsom, England, cat. no.AB-0366).21. Size marker Φ×174/HaeIII (0.1 mg/mL, Promega, Southampton, England,cat. no. G1761). Add 450 µL of ddH 2 O to vial of the size marker. Add 20 µL of