In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1.
In this report we describe a coliphage lambda vector system for cloning endo R. EcoRI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing lambda DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For lambda wild-type DNA the efficiency of in vitro packaging (10(6) to 10(7) plaques produced per microgram of added DNA) is equal to, or better than, the standard CaCl2 transfection method. The use of a Dam mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 and R.EcoRI fragments were cloned.
D003090:Coliphages / Q000235:genetics*; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004279:DNA, Viral; D004926:Escherichia coli / Q000235:genetics*; D014162:Transfection
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes