Large-scale preparation of a DNA fragment containing the strong promoter A1 of the phage T7.
A procedure has been developed to isolate DNA fragments on a large scale. A DNA fragment of 130 base-pairs containing the strong promoter A1 of the phage T7 was purified to homogeneity in amounts of 10 mg. The procedure includes the rapid purification of gram amounts of plasmid DNA, a new, simple method to separate small DNA fragments from the vector by a phenol/water partitioning system, and a liquid-liquid PEG-dextran partition chromatography for the final purification of the fragment. The fragment was cloned in two vector systems: The vector pDS1, to1+ (1), containing an efficient terminator downstream from the promoter integration site, gives high yields, 3-4 mg plasmid DNA per liter medium. In the plasmid pWH802 (2), which is not specially designed for the amplification of a strong promoter, the integration of the promoter was possible but the yield decreased by a factor of about 50. The stability of the inserts was tested in both systems. Monomeric inserts were stable in both plasmids, multimeric inserts up to a tetramer were only stable in pWH802. Only one orientation of the fragment was found.
D001709:Biotechnology / Q000379:methods; D002845:Chromatography; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004279:DNA, Viral / Q000302:isolation & purification*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis
D004279:DNA, Viral; D011506:Proteins; D012313:RNA