Hybridization cassettes

Special hybridization ovens or convenient acrylic cassettes to be placed in a waterbath are commercially available. The membrane is placed in the hybridization cassette (e.g., from Bios Corp., New Haven, CT 06511 Fax (203) 773-0017), covered with (pre)hybridiza-tion solution, the top closed over a cover and incubated at the desired temperature. Hybriboxes can also be constructed as reported by Cohen et at. (1990 Section 7.2.6). The hybridization... 

Before prehybridization, nitrocellulose should be wetted with 1 X SSPE (or 1 X SSC), containing 0.1% SDS, whereas nylon membranes can be used directly. Prehybridization and hybridization is performed in polythene bags, plastic boxes (hybridization cassette) or hybridization incubators (Section 8.1.2). The cassettes and incubators are economical in the use of hybridization solutions. For plastic bags, use 0.25 ml/cm for nylon membranes and 0.1 ml/cm for nitrocellulose membranes of prefiltered (important ) prehybridization solutions. The bags should be massaged to completely distribute the solution and incubated with agitation. 

Rehydration without prehybridization Place array slide into hybridization cassette and put 15 pL of water in each end of the chamber to ensure high humidity. Seal the chamber and place in oven at 42°C for 30-60 min. 

Bouin s solution is one of the traditional ways to harden cell pellet. Some cytologists believe it provides the best cellular details, especially nuclear features in cell blocks.28 The major steps are (1) After centrifugation, fix the cell pellet with Bouin s solution. (2) After 2h, discard the solution. (3) Remove the hardened cell pellet from the tube, wrap it with lens paper, and transfer it into a cassette for further processing. We have been using this method for many years. In our experience, most of the time, ICC results are consistent with IHC from the surgical specimen. The biggest drawback of this method is the toxicity of Bouin s fixative which creates biohazard and safety issues for the laboratory. We also found cell blocks gave poor fluorescence in situ hybridization (FISH) results after Bouin s fixation. 

Naked plasmid DNA was not only trapped in the cytoplasm around the site of microinjection, as visualized by fluorescence in situ hybridization (FISH) or using FITC-labeled DNA, but was also eliminated rapidly at physiological temperature (Lechardeur et al., 1999). The disposal of the DNA was completely prevented when the cells were kept at 4°C (Lechardeur et al., 1999). A similar conclusion was reached by monitoring the amount of microinjected expression cassette by the polymerase chain reaction (PCR) (Pollard et al., 2001), suggesting that the metabolic instability of naked DNA contributes to the low efficiency of gene transfer (Lechardeur et al., 1999 Mirzayans et al, 1992 Neves etal., 1999 Pollard et al., 2001). 

A microfluidic device has been developed, which allows hybridization in 15 min and was able to discriminate between four Staphylococcus strains (134). A similar device has also been developed for cell lysis (135). The goal of these devices is to allow a clinical lab to take a patient specimen dispense it into a cassette and have DNA extraction, labeling, hybridization, and scanning occur automatically (136). 

PFO puruvateiferredoxin oxidoreductase, HCP hybrid cluster protein, PNO pyruvate NADH oxidoreductase, Nbp35 P-loop NTPase, Nar/Narf hydrogenase-like protein, Rli ATP-binding cassette protein... 

For expression of the polyphenolic protein, the coding sequence is inserted into an expression cassette composed of a promoter, translation initiation sequence, signal sequence, and transcription terminator. The promoter sequence in YpGX285 is derived from two yeast genes, GAL1 (32) and alpha factor (MF-al) (25). This hybrid promoter provides for efficient, regulated, transcription of... 

Fig. 4.2 Detection of homologous recombinants by Southern blot. Restriction enzyme A cuts once in the homologous side arms of the targeting construct and once within the neomycin selection cassette. For Southern blot, the genomic DNA is digested with restriction enzyme A and hybridized with an external probe which stains bands of different size for homologous recombinants or the wild-type gene. In case of heterologous recombi...

The visualization of hybridization between the RNA probe and the DNA fragments on the blot is based on an enzyme-linked immunoassay. An antidigoxigenin/alkaline phosphatase conjugate is bound to the digoxigenin component of the probe and then incubated with the substrates X-phosphate and nitroblue tetrazolium (see Section 2.) under alkaline conditions, which will result in purple-blue precipitates on the membrane within a couple of hours. Color detection thus saves time and money as it does not require X-ray films, cassettes, and intensifier screens. For a permanent record the result can be photocopied or photographed. 

Early investigation of Li-air batteries showed that the air cathode was the most important challenge for their development, although lithium anode side needs another challenging endeavor in the hybrid configuration. Prabaharan et al recently reported on their proprietary lithium metal/SE monolithic cassette as the protected lithium anode. ... 

It IS useful to first place the hybridized slides in an autoradiography cassette and expose them against a high resolution X-ray film for 1-3 d at room temperature. This type of autoradiography is insufficient for analytical purposes. 

S.S. Chandran, H.G. Menzella, J.R. Carney, D.V. Santi, Activating hybrid modular interfaces in synthetic polyketide synthases by cassette replacement of ketosynthase domains. Chem. Biol. 13, 469 74 (2006)... 

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