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Summary: Auble Lab
In Vitro NER Assay
Reagents:
Water
YPD
Yeast extraction Buffer (200 ml): 0.2 M Tris-acetate (pH 7.5) (40 ml), 0.39 M
(NH4)2S04 (78 ml), 10 mM MgSO4 (2 ml), 20% Glycerol (40 ml), 1mM
EDTA (pH8.0) (0.4 ml), and water (39.6 ml). Store at 4°C. Add DTT to
0.1 mM and protease inhibitors before use.
Protease Inhibitor Stocks: 100X PMSF (100 mM in ethanol); 1000X
Benzamidine hydrochloride (Ben) (1M in water); 1000X Pepstatin A (Pep)
(3.5 mg/ml in DMSO); 1000X Leupeptin (Leu) (1mg/ml in water); 200X
Aprotinin (Apo) (2 mg/ml in water). Store all stocks at -20°C.
Solid Ammonium Sulfate
Dialysis Buffer (500 ml): 20 mM HEPES-KOH (pH 7.5) (10 ml), 20% Glycerol
(100 ml), 10 mM MgSO4 (5 ml), 10 mM EGTA (10 ml), and water (367.5
ml). Store at 4°C. Add DTT to 5mM and 100X PMSF before use.
Plasmid DNA: pGEM-3Z (2.8 kbp) and pGEM-3Zf(-) (3.2 kbp). Purified by
Qiagen DNA purification kit.
N-acetoxy-2-acetylaminofluorene (NAAAF) 1 mM in ethanol. Store at -20°C.
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