Colony hybridization membranes

Plasmids are also encountered in eukaryotes, although less commonly than in bacteria. Examples are the 2- x circle in yeast, the senescence (aging) plasmids in the fungus Neurospora and the plasmids associated with male sterility in corn. In addition, there are many other extrachromosomal genomes such as those from mitochondria, chloroplasts and viruses. In addition, colony hybridization type experiments can be carried out on single genes or on transcripts and may resemble in situ hybridization, except for the transfer to membranes. 

The bacterial colony hybridization technique can be adapted easily to eukaryotic cells (Avraham et al., 1989). Nitrocellulose membranes, autoclaved for 30 min, are placed in a sterile plate containing sterile PBS and the monolayer cultures are washed with sterile PBS twice (PBS is Ca and Mg free). Three drops of PBS are added to each plate and the membrane is placed on top of the cells and left for 2 min (no air bubbles both membrane and plate marked). The membranes are then gently lifted and fresh medium is added to the cells to regenerate in a 37 C incubator. 

DNA hybridization assays rely on the properties of single strand DNA to hybridize with complementary sequences. They also use the property of DNA to bind to nitrocellulose membranes (Bouvet and Vemozy-Rozand 2000). The availability of cloned stxl and stx2 genes allow the development of DNA probes for the detection of STEC. Initially, probes labeled with or were used to screen a large number of E. coli fecal isolates by colony hybridization. These techniques were specific and sensitive and could differentiate between stxl and stx2 if stringent conditions were used. 

Carefully lift up the membrane with two forceps. Autoclave the membrane for 1 min in dry cycle to denature the bacterial DNA. Cross-link DNA on the membrane by using a UV crosslinker and perform colony hybridization. 

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). 

Place the membrane, numbered side down, on agar as in step B5 and leave for 10 min. Punch the membrane and agar as in step B5 and peel off the membrane with blunt tweezers. This membrane either serves directly for hybridization or is placed on an agar plate to obtain larger colonies or on a plate containing chloramphenicol (200 p.g/ml) to obtain an amplification of vector or to obtain more replicas. 

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. 

Each sample was diluted in decimal stages to ensure the reliability of the end result. The dilute solutions were seeded evenly on the surface of the Petri dishes with sterile beads. This technique is necessary if the cells are to be identified by DNA/DNA hybridization (Volume 1, Section 4.3.5) on colonies or PCR (Volume 1, Section 4.3.6). If the wine contains very few microorganisms, it is filtered onto a 0.45-tim membrane, which is then deposited on the specific... 

Probes labeled directly with HRP have been used in many membrane hybridization applications (5,6), including Southern blots. Northern blots, colony and plaque screening, PCR product detection/ identification, YAC clone screening, and RFLP analysis. 

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