By Ray H. Gavin
Ray H. Gavin brings jointly a world panel of skilled researchers to aspect the comfortably reproducible equipment that make the most of biochemistry, immunology, genetics, microscopy, and snapshot research for investigating cytoskeleton constitution and serve as. each one protocol includes confirmed step by step directions that allow either the beginner and the skilled researcher to accomplish winning experimental effects. The protocols make the most of assorted version structures in numerous organisms, together with Saccharomyces, Micrasterias, Tetrahymena, Drosophila, Spisula, and Xenopus. Microscopy functions contain digital-video microscopy and computer-assisted platforms for the assessment of phone motility and morphology. cutting-edge and hugely useful, Cytoskeleton equipment and Protocols makes to be had a various selection of robust experimental platforms and instruments for effectively learning cytoskeleton constitution and serve as.
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Extra resources for Cytoskeleton Methods and Protocols (Methods in Molecular Biology Vol 161)
2) PAGTGKT (primer S2); (3) CFDEFNR (primer A2); (4) FITMNPG (primer A1); and (5) HYDFGLRA (primer A3). We have utilized primers based on the universal genetic code as well as biasing the sequences according to Tetrahymena codon usage (18); we have detected little difference between primers derived by the two methods. The primers listed in this chapter are based on the universal genetic code. Here we describe PCR amplification with the S2 and A2 primers. At the 5' end of S2 is an added BamHI site; at the 5' end of A2 is an added EcoRI site.
Denhardt’s solution (50X): 500 mL: 5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin. 6. 15M NaCl, 2 mM EDTA, 30 mM Tris-HCl, pH 8. 8. Electrophoresis 1. 5X TBE. 2. Sequencing gel solution: 7% acrylamide/bisacrylamide, 8M urea. 3. 1. Culturing Cells 1. CultureTetrahymena thermophila strain B2086 cells in Neff’s medium at 30°C on a shaker. Harvest exponentially growing cells at a density of 3–5 × 105 cells/mL. Dynein Heavy Chain Gene Family 21 Collect cells by centrifugation at 1500 rpm for 1 min at room temperature in pearshaped oil tubes (HN-S II centrifuge, International Equipment Company).
3. 1. 1. Videomicroscopy The two dimensional dynamics image analysis system, 2D-DIAS, is diagrammed in Fig. 1. For the 2D analysis of cell behavior in the absence of chemoattractant, samples are inoculated into a perfusion chamber, and allowed to adhere to the wall of the chamber for 5 min. The chamber is perfused with buffer or media at a rate of 4 mL per min in order to continuously refresh the environment (16). Images are videorecorded onto half inch or three quarter inch videotape, or digitized directly, for at least 10 min at 25× magnification using brightfield optics.