Recombination plaque purification

RNA probes, and the resulting film exposures can be used to locate the recombinant X plaque responsible for a specific hybridization signal. After a series of plaque purification steps (repeated plating and plaque isolation), an individual X recombinant clone can be purified to homogeneity. 

Viral titer determination and plaque purification of the recombinant viruses are usually not necessary. Successful infection and production can be monitored by fluorescence microscopy. 

Alternative methods have been developed to obtain virus stocks without plaque purification for expression of recombinant proteins in infected insect cells. The Bac-to-Bac technology (Invitrogen, Thermo Fisher Scientific) avoids homologous recombination in insect cells by using site-specific transposition in E. coli. With this technology, recombinant bacmid-DNA is generated that is used to transfect insect cells. 

Figure 2. Screening of a pea cDNA library for CA immunopositive clones. Nitrocellulose filters prepared from 86 mm plates containing approximately 10,000 recombinant plaques were incubated with the CA antisera. Specifically bound antibody was detected with horseradish peroxidase conjugated goat anti-rabbit IgG. A positive signal on a primary plate is shown (A) as well as the tertiary stage of plaque purification (B). A control filter with no positive signals is also presented (C).

This procedure has been adapted to the AMA cell line but vaccinia virus will infect most cell lines ranging from Drosophila Scheider (S3) cells to different human cell lines, but it does so with very different efficiency and the procedures must be adapted to the cell lines used. The virus infects most epithelial cell lines efficiently but grows poorly on fibroblasts. As AMA and most other cell lines are not TK cell lines, we only apply the positive selection (for the presence of the gpt gene). The procedure for BrdU selection is described in Mackett et al. (1985). Also, the intent of this procedure is to link a specific cDNA to a spot on a two-dimensional gel and this does not require plaque purification. If the aim is to do functional studies of a protein it may be advisable to do a plaque purification, as there can be some nonrecombinant viruses left over and the yield of recombinant protein may be increased if they are removed. 

Protocol 1.8 Purification of recombinant virus and determination of viral titre by plaque assay... 

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