Aging Aging

100 Engel, Adibzadeh, and Pawelec5. QIAquick® Gel Extraction kit (Qiagen, Hilden).6. Rapid DNA Ligation Kit (Boehringer Mannheim).7. RAP-PCR Kit (Stratagene, Heidelberg).8. RNeasy Mini Kit (Qiagen, Hilden).2.6. Molecular Weight Markers1. 1 kb DNA Ladder (Gibco-BRL, Eggenstein).2. 100-basepair DNA Ladder (Gibco-BRL, Eggenstein).2.7. Silver Staining1. Fixation solution: 10% acetic acid or 15% EtOH/5% acetic acid. Prepare a freshsolution prior to use.2. Silver reacting solution: 0.1% AgNO 3 , 200 µL of formaldehyde (37%). Prepare afresh solution prior to use.3. Developing solution: 2.5% Na 2 CO 3 , 200 µL of formaldehyde (37%), 200 µL ofNa-thiosulfate (2%). Prepare a fresh solution prior to use.4. Stopping/desilver solution: 2.0% (w/v) Glycine. Prepare a fresh solution priorto use.5. Impregnating solution: 10% Glycerol. Prepare a fresh solution prior to use.2.8. Cloning of DNA Fragments1. 50 mM CaCl 2 . Store at 4°C.2. 10 × Klenow buffer: 500 mM Tris-HCl, pH 7.5, 60 mM MgCl 2 , 10 mM dithiothreitol(DTT). Freeze until use, then store at 4°C.2.9. Bacterial Growth Media1. LB Medium: 10 g/L of bacto-tryptone, 10 g/L of NaCl, 5 g/L of glucose, 5 g/L ofyeast extract. Store at 4°C.2. LB plate agar: LB medium, 15 g/L of agar. Store at 4°C, cover down.2.10. Plasmid Isolation1. Resuspension solution: 50 mM Tris-HCl, pH 7.5; 10 mM EDTA. Store at roomtemperature.2. Lysis solution: 0.2 M NaOH, 1% sodium dodecyl sulfate (SDS). Store at roomtemperature.3. Neutralization solution: 2.55 M K-acetate, pH 4.8. Store at room temperature.2.11. Northern Blotting1. 20× SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0 (adjust with NaOH). Store atroom temperature.2. Hybridization buffer: 1 mM EDTA, pH 8.0, 0.25 M Na 2 HPO 4 , pH 7.2, 7% SDS,1% BSA. Can be stored at 4°C for a few days.3. Washing buffer: 1 mM EDTA, pH 8.0, 20 mM Na 2 HPO 4 , pH 7.2, 1% SDS. Storeat room temperature.