Immobilization baking

After activation by heating, the catalyst was dusted over the surface of a thin polydimethylsiloxane (PDMS) layer, being coated on the PDMS top plate of the micro reactor [19]. Such a modified plate was baked for 1 h at 100 °C. A high surface area and firm immobilization of the catalyst resulted. Then, the micro reactor was assembled from the top and another bottom plate, having at one micro-channel wall the catalyst layer. Stable operation with the PDMS micro reactor up to 175 °C could be confirmed. 

A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper, which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. 

Hybridization can be performed by merely spotting the sample to a membrane, where it is immobilized by baking and subsequently hybridized to a suitable probe. Sample application can be performed with commercially available manifolds that apply sample into multiple wells of the manifold and let sample migrate as spots or slots into the membrane hence the name dot blot or slot blot hybridization (W2). The sample wells are repeatedly washed prior to removing the membrane to bake or irradiate in order to fix the sample, which is then ready for hybridization with probe. 

Disassemble the apparatus when wells are empty and UV irradiate the samples (better and more convenient than baking). Baking or UV irradiation of alkaline-immobilized DNA on charged nylon membranes may be counterproductive. When using UV irradiation for the first time, it is recommended to optimize the time required (usually 1-5 min) for best binding to the damp membranes (e.g., uncovering a successive dot every 30 s) always use the same distance from the source. Store as in A7. 

Fast blot methods to minimize nucleic acid extraction and immobilization steps have been developed. Those with nylon as a solid phase can take advantage of the ability of NaOH to dissociate cells, denature DNA and immobilize DNA. Nitrocellulose membranes have a lower binding capacity and co-immobilization of nucleic acid and protein from neutral solutions can be a problem. Bresser et al. (1983) used hot concentrated Nal to inhibit protein immobilization, to denature DNA and to irreversibly bind the nucleic acid to nitrocellulose (no baking required). This method can also be used for RNA. About 10 cells are minimally required for a unique DNA sequence, whereas > 0.01% of total mRNA can be detected by the Nal methods. 

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