Colony hybridization master plates

A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper, which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. 

Invert the filter and gently lay it onto fresh media just prior to lysis to create a replica of the colony pattern of the filter. (Mark the plate to indicate the orientation of the filter on it) After an appropriate incubation, this becomes a master plate from which viable analogues of desirable colonies, as identified on the filter after hybridization and processing, can be recovered... 

In another approach, the colony hybridization technique (Figure 18D, bacteria are screened by using a radioactively labeled nucleic acid probe, an RNA molecule or a single-stranded DNA molecule with a sequence complementary to that of a specific sequence within the recombinant DNA. Bacterial cells are plated out onto solid media in petri dishes and allowed to grow into colonies. Each plate is then blotted with a nitrocellulose filter. (Most of the original colonies remain on the petri dishes.) The cells on the nitrocellulose filter are lysed, and the released DNA is treated so that hybridization with the probe can occur. Once nonhybridized probe molecules have been washed away, autoradiography (Biochemical Methods 2.1) is used to identify the colonies on the master plate that possess the recombinant DNA. 

Bacterial cells are plated onto a solid medium that only allows the growth of transformed cells. Once the colonies become visible, the plate is blotted with a nitrocellulose filter. The cells clinging to the filter are lysed and the released DNA is denatured and deproteinized. After a labeled probe is added, unhybridized probe molecules are washed away. Cells that possess DNA sequences that hybridize with the probe are identified by comparing the autoradiogram of the filter with the master plate. 

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