with PEI, one can use commercially available plasmids [e.g., p cytomegalovirus (CMV)-luciferase from Promega; pCMV(nls)-Lac-Z from CLONTECH (Montigny-le-Bretonneux, France)]. For CAT, the most efficient construct we have tested is pcis-CMVCAT provided by R. Debs and coworkers (13).
verifying plasmids.
EDTA (TAE) or Tris-borate EDTA (TBE), see Reference 10].
of DNA concentrations (OD260) and purity of DNA (OD260/OD280 >1.8).
1. For plasmid DNA preparation and purification we recommend use of Jetstar columns (GENOMED, Raleigh, NC, USA). The system is based on anion exchange columns. According to the manufacturer’s instructions and solutions supplied, bacteria resulting from maxiculture are lysed by alcali. Large membrane debris are eliminated using potassium acetate and centrifugation. The resulting supernatant is loaded on columns, and DNA is eluted with a solution containing approximately 2 M NaCl, then centrifuged after isopropanol precipitation. The pellet is washed with ethanol 70% (-20°C) and resuspended in water or Tris-EDTA (TE) at high DNA concentration (≥0.5 μg/μL in TE). This is to ensure that when diluting DNA to its working concentration (≤0.5 μg/μL in 5% glucose), the final TE concentration is not greater than 1 mM Tris/0.1 mM EDTA (standard TE/10). These plasmid preparations are endotoxin free. 2. One microliter of each DNA preparation is diluted in 1 mL of sterile water and analyzed at 260 and 280 nm. According to Sambrook et al. (10), 1 U OD260 correspond to 50 μg/mL of double-stranded DNA. 3. Agarose gel electrophoresis is used to verify that the plasmid DNA is not denatured, is free of RNA, and is mainly supercoiled. Restriction map analysis can be used to check constructions at this point. To this end, native and digested plasmids are analyzed using 0.8% agarose gel electrophoresis in TAE, with bromophenol-blue and a DNA molecular weight marker (10). The gel is observed on a UV transilluminator (312 nm) and photographed with a Sony video equipment from OSI (Maurepas, France).
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