By Ray H. Gavin
Within the ten years because the booklet of the 1st variation, nice advances in fluorescent labeling, optics, and pattern instruction have considerably superior the imaging power of microscopy, bearing in mind a continuous refinement of our realizing of the cytoskeleton as a dynamic synergy of parts. In Cytoskeleton tools and Protocols, moment variation, across the world well known specialists current ideas which replicate the various contemporary technological advances in experimental instruments for cytoskeleton learn with emphasis on animal, plant, protist, and fungal version platforms. This state-of-the-art quantity comprises equipment for live-cell imaging, fluorescence microscopy, electron microscopy, research of telephone and organelle motility, isolation of cytoskeleton parts, and proteomics, among different issues. As a quantity within the hugely profitable tools in Molecular Biology™ sequence, chapters comprise introductions to their respective matters, lists of the required fabrics and reagents, step by step, without problems reproducible laboratory protocols, and notes that offer unpublished technical info on troubleshooting and keeping off recognized pitfalls. updated and finished, Cytoskeleton tools and Protocols, moment variation serves as an incredible advisor to scientists who desire to proceed this fruitful and significant organic study.
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Additional resources for Cytoskeleton Methods and Protocols, 2nd Edition (Methods in Molecular Biology Vol 586)
200 mg/L G418 for kanMX6; see Note 7). Incubate 2–3 days, and replica-plate onto a fresh plate containing the drug. Colonies can be picked from this plate for further analysis. 4. PCR Screening, Protein Expression, and Functional Analysis PCR is used to validate insertion of the tag into the target locus. This analysis uses genomic DNA isolated from transformants as a template and a pair of primers (20–25 bases) that hybridize on both sides of the insertion. Optional PCR can be carried out using a third primer that anneals to a region within the integration cassette.
Traditionally, imaging of actin filaments or microtubules in cultured cells was accomplished by microinjection of actin or tubulin protein labeled with small fluorescent molecules (11). , eGFP or mCherry) fused to actin or tubulin has permitted generation of stable cell lines expressing these proteins by transfection with the relevant DNA (12). However, we have found that injection of fluorescent actin or tubulin protein into embryos can be less than satisfactory, for two reasons. First, the small volumes required (5 nL or less) means that extremely high protein concentrations are required.
3. Microinjection of Xenopus laevis Embryos We microinject Xenopus embryos at the 1-, 2-, 4- or 8-cell stage using a PLI-100 picoinjector (Harvard Apparatus; see Note 5). 1). , morpholino) see Note 6. When injecting RNA, we use measures to limit RNases by cleaning injection areas with RNaseZAP® (Ambion), using RNase-free reagents and by wearing gloves at all times. 1. 5 h after fertilization (at 17°C). 1. Pull needles using 10 µL glass capillaries and a micropipette puller. 2. Using dissecting microscope and sharp forceps, cut off the end of the needle to produce a point approximately 5–10 µm in diameter.