Amicon ultra 30k centrifugal filters

Genomic DNA was isolated from blood with Gentra Puregene Blood kit (Qiagen, cat no. 158389) according to the manufacturer’s instructions. DNA (7.5 μg) was digested with 3 U/μg DNA Mbo I (10 U/μl, Thermo Scientific, cat no. ER0812) at 37°C for 1 h. The DNA was purified with Amicon Ultra 30K centrifugal filters (Millipore, cat no. UFC503024) and divided into 1 μg aliquots. Treatment of DNA with cisplatin (cis-Diamminedichloroplatinum(II)) was done according to Murray et al. (24 (link)). In a total volume of 100 μl, 1 μg DNA was incubated in 25 mM Tris–HCl, pH 7.8 with 1–15 μM of cisplatin (Sigma-Aldrich, cat no. 479306), at 37°C for 18 h in the dark. The DNA was ethanol precipitated with sodium acetate and resuspended in 3 μl TE buffer (10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0). For denaturation experiments, 1 μg human genomic DNA, in 3 μl TE, was heated up at 94°C for 4 min. Plasmid pUC 18 was digested with Hinf I (Thermo Scientific, cat no. ER0801) and Ssp I (Thermo Scientific, cat no. ER0771) 1.5 U enzyme per microgram DNA, prior to cisplatin treatment.

E. coli C41(DE3) with plasmid constructions pET29c::Ma1, pET29c::Ma2, pET29c::Ab and pET29c::tDGAT were grown in 2 l flasks containing 1 l LB supplemented with 50 ug/ml kanamycin sulfate and induced with 1 mM IPTG when the OD_600nm was approximately 0.6. Cell cultures were collected 3 hours later by centrifuging in a JA10 rotor (Beckman Coulter, Brea, CA) at 4,400 g for 15 minutes a 4°C and stored frozen at -20°C until analyzed. The supernatants were discarded and the pellets were resuspended in buffer A (NaCl 1 M, TrisHCl 50 mM pH 7.5 and PMSF 100 μl), sonicated and clarified by ultracentrifugation at 100,000 g for 15 minutes at 4°C. Supernatants were loaded onto a Níckel HisTrap HP column (GE Healthcare, 5 ml) previously equilibrated with buffer A. The His-tagged bound proteins were eluted by a gradient of concentration of imidazole (0-300mM; Sigma, St. Louis, MO). In order to improve the purification, the eluted protein was concentrated using Amicon Ultra 30k Centrifugal filters (Millipore, Ireland) and loaded into a size-exclusion chromatography Superdex 75 GL10_30 column (Amersham Pharmacia Biotech, UK) previously equilibrated with buffer B (NaCl 150 mM TrisHCl 50 mM pH 7.5 1 mM EDTA). Purified proteins were stored at -80°C in glycerol 20% for further experiments.