Plaque hybridization

After Southern transfer of restriction-digested genomic DNA, filters are prehybridized for 1-4 hr. The probe is boiled for 10 min to denature it, added to fresh hybridization buffer, and hybridized overnight. To calculate appropriate hybridization temperatures in buffers with or without forma-mide, and for details of hybridization buffers and washes, see standard protocols.13 For hybridizations at low stringency to detect signals from fragments of approximately 65-70% overall sequence similarity, we apply 55° for hybridization and washes in 6X SSC without formamide. Similar protocols can be used for colony and plaque hybridizations. 

Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-... 

A survey on molecular biology products (Ausubel et at, 1991) indicated that for nitrocellulose about 70% of respondents preferred S S NC (other manufacturers < 10%) but that the preference for nylon membranes differed little among Schleicher Schuell, Dupont NEN and Amersham. In the same survey, respondents had no clear preference for either nylon or nitrocellulose for Southern hybridization but a clear preference for nitrocellulose in the case of colony/plaque hybridization (where concentration of target is sufficient highly but background should be suppressed). 

Dilute phage stock to =200 plaques/0.1 ml and replate (Table 4.7). Repeat plaque hybridization and pick well-isolated plaques. 

Despite the wide use of radioprobes in colony or plaque hybridization assays, nonradioactive probes can be advantageous. The use of biotinylated probes, initially the most common among nonradioactive detection systems, is limited since biotin-streptavidin systems tend to give high background levels with bacterial material unless specific measures are taken. The more recently developed DIG (Table 7.2), but also other hapten-antibody systems such as sulfonated probes, are very attractive alternatives. The main restriction is that monoclonal antibodies (commercially available) should be used since polyclonal antisera often contain antibodies against bacteria. The main drawback of nonradioactive probes is the ability to reprobe the same membrane. It is possible, however, to strip a membrane of its probe after a colorimetric detection and to perform a chemiluminescent detection or vice versa. 

K9. Kincaid, R. L., and Nightingale, M. S., A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling. BioTechniques 6, 42-49 (1988). 

RNA) (colony or phage plaque hybridization similar clone identification) Northern ... 

Clones containing genomic DNA of a particular sequence may be identified by plaque hybridization, which is similar in principle to colony hybridization for detection of particular plasmid recombinants. A lawn of E. coli containing several thousand plaques of independent recombinants is blotted on to a nitrocellulose filter. The DNA is released from the blotted recombinant phage, denatured by alkali, and neutralized. A radioactive probe of nucleic acid containing sequences of the gene of interest is then hybridized to the DNA plaques on the filter. Positive plaques, detected by autoradiography as radioactive DNA-RNA or DNA—DNA hybrids, are then picked off and amplified at lower plaque density (approx. 100 per plate). 

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