Screening of cloned recombinant DNA in bacteria by in situ colony hybridization.
We have developped in situ methods of colony hybridization in which there is no need to replicate colonies one by one prior to hybridization. The best method consists in promoting partial lysis of the colonies on the plates by means of a resident thermoinducible prophage. It appears that colonies are heterogeneous with respect to prophage induction, so that survivors remain in each colony. Blotting onto nitrocellulose filters and hybridization with a highly radioactive probe permits the screening of many thousands of colonies per plate for the presence of a DNA sequence carried by a plasmid and complementary to the probe. This procedure greatly facilitates the isolation of recombinant plasmids which carry a specific DNA sequence. We also describe a second, less efficient procedure which does not use prophage induced lysis, and is potentially usable with B2 or EK2 safety systems, without modification of the bacterial hosts.
D003090:Coliphages / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005374:Filtration / Q000295:instrumentation; D008242:Lysogeny; D008722:Methods; D009693:Nucleic Acid Hybridization; D010957:Plasmids
D004274:DNA, Recombinant