Plating Protocols

Many plating protocols advocate the use of a flash step where a significantly higher overpotential is applied to ensure that the entire substrate is covered with metal before the potential is reduced to the plating potential. This has been shown to be effective in ionic liquid and significantly improves the corrosion resistance of the coatings [7]. 

In this chapter we would like to present some plating protocols for the electrodeposition of aluminum, lithium, tantalum and zinc from different ionic liquids. These recipes have been elaborated in our laboratories and should allow the beginner to perform his first electrodeposition experiments. For aluminum we give four different recipes in order to show that the ionic liquid itself can strongly influence the deposition of metals. In the case of tantalum the deposition of the metallic phase is not straightforward as, in unstirred solutions, the more nonstoichiometric tantalum halides form the higher the current density for electrodeposition. Apart from the zinc deposition all experiments should be performed at least under dry air. 

This procedure, using microwells, is simple. Culture samples are counted and diluted to the desired concentrations (the PE dilution is best made from the PEstg dilution before adding the 6TG), selective chemicals are added to the appropriate diluted samples, and the samples are apportioned over the microwell plates. The plates are covered and placed in a 37°C, humidified, 5% CO2 incubator macroscopic colonies appear and are counted by 10-12 days. Readers who cannot handle the Poisson distributions should refer to a description of standard agar-plating protocol and are condemned to years of tedious labor,although feeder layers need not be used. 

Another approach, the Single Plate Protocol (available at http //www. nuigalway.ie/microbiology/prof peter smith.html) that might be suitable for laboratories that analyse only a small number of clinical isolates per year has been proposed. In this protocol the susceptibility of a clinical... 

The type of plate, chamber system, composition of mobile phase, running time and detection reagent used must naturally all be recorded The sample protocol illustrated in Figure 62 can be employed... 

Figure 4.4 The general protocol for information extraction from an herbal text (A-E) is paired with case examples from our work with the Ambonese Herbal by Rumphius. (A) Text is digitized. (B) Through either manual reading or automated extraction the plant name(s), plant part(s), and symptoms or disorders are identified. (C) These extracted data are then updated (as necessary) to reflect current names of the plants, using the International Plant Names Index (IPNI), and the pharmacological function(s) of the described medicinal plants are extrapolated from the mentioned symptoms and disorders. (D) The current botanical names are queried against a natural products database such as the NAPRALERT database to determine whether the plant has been previously examined. (E) Differential tables are generated that separate the plants examined in the literature from plants that may warrant further examination for bioactivity. (Adapted from Trends in Pharmacological Sciences, with permission.) See color plate.

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

In addition, it was assumed that too high reactivation temperatures were used, as a comparison with literature protocols reveals [60, 62]. Cracks in the plate and void areas with catalyst loss seem to corroborate this assumption. 

In most cases, in consultation with the HTS group, the research area laboratory will develop a benchtop assay that is at least compatible with the HTS format of choice for their target. This tends to facilitate project transitions and provides a tool that the research area laboratory will use later to follow up hits and develop SAR. In other cases, the HTS assay development group will assume all responsibility for assay development. The formality of the transfer of the project from the research area to the HTS group varies between organizations, but the outcomes are quite similar. All of the details of the prototype assay are reviewed by both teams, and, where applicable, reagents, protocols, and even plates or pipette tips are exchanged. 

After an hour of incubation, the medium is replaced with Neurobasal culture medium. Add 2, 4, and 10 ml of Neurobasal culture medium to the 35, 60, and 100-mm plates, respectively. After 5 to 7 days in culture, the medium must be half-changed, that is, about half of the old medium is replaced with the same volume of fresh Neurobasal culture medium. Halfchange the medium every 5 to 7 days until the neurons are ready to use. For neurons younger than DIV (days in vitro) 7, if necessary, the medium can be completely replaced without affecting cell viability. In contrast to Brewer s protocol (Brewer et ai, 1993), we do not include 25 /iM glutamate during the first four days of culture and find no adverse effect on neurons. 

A general flowchart is presented in Fig. 13.5B that we followed for identifying and sorting inhibitors of translation. Shown below is an in vitro translation protocol tailored for ten 96-well assay plates (800 compounds), which can be scaled up or down as required. Negative and positive controls are present in wells A1 to D1 and El to HI, respectively. Compounds are added to wells A2 to Hll. Column 12 is left blank and could be used for additional controls, if desired. 

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