By Peter Satir, Søren Tvorup Christensen
This quantity is the results of an explosion of molecular-based examine on Cilia, which started with the invention of the universality of intraflagellar shipping (IFT) and ciliary genomics/proteomics. The chapters during this e-book conceal issues corresponding to: excessive solution imaging and practical characterization of sensory and first cilia in mammalian cells and zebrafish, how to research ciliary-mediated chemoresponse in Paramecium, and strategies to review centrosomes and cilia in C. elegans and Drosophila. Written within the hugely profitable equipment in Molecular Biology sequence structure, chapters contain creation to their respective themes, lists of the mandatory fabrics and reagents, step by step, with ease reproducible laboratory protocols, and tips about troubleshooting and heading off identified pitfalls.
Practical and state-of-the-art, Cilia: tools and Protocols is vast and covers motile, sensory, and first cells. it's a necessary source to someone attracted to getting into the sphere of ciliary biology utilizing version organisms, together with flagellate algae, ciliates, planaria, nematodes, bugs, zebrafish, and mammalian cells.
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Additional resources for Cilia: Methods and Protocols
Wash cells in ice-cold PBS and add boiling lysis buffer to the Petri dish. 2. Scrape cells off using a rubber policeman and transfer the cell lysates to microcentrifuge tubes and keep the samples on ice. 3. Sonicate the cell lysates for 3 × 10 s. Rest on ice in between to avoid heating of the samples. 4. Centrifuge at 20,000 × g for 20 min to precipitate the cell debris. Transfer the supernatant to new microcentrifuge tubes and keep them on ice. 5. , the Bio-Rad DC Protein Assay. 30 Patricia Verdier et al.
Incubate for 2 min at 25 °C. 6. Add 2 μl SuperScript II reverse transcriptase enzyme to the tube and incubate for 10 min at 25 °C, followed by 50 min incubation at 42 °C and 15 min at 70 °C to inactivate the enzyme. 7. Store the cDNA at −20 °C until use. 2 PCR and Cloning Procedures 1. 5 μl. The primers used in this study are shown in Table 1 and a schematic overview of the fusion proteins generated is shown in Fig. 1. 2. 5 μl cDNA from RPE1 cells (25 ng/μl) and 1 μl Herculase II Fusion DNA polymerase.
1007/978-1-4939-3789-9_2, © Springer Science+Business Media New York 2016 15 16 Patricia Verdier et al. GFP hTERT-RPE1 IFM LB LDS MFI MSP NPHP4 OCRL PACT PBS RT siRNA TBS-T TPR TRAPP TRAPPC 1 Green fluorescent protein Human telomerase-immortalized retinal pigmented epithelial Immunofluorescence microscopy Luria-Bertani Loading sample buffer Mean fluorescence intensity Major sperm protein Nephronophthisis 4 Oculocerebrorenal syndrome of Lowe Pericentrin-AKAP450 centrosomal targeting Phosphate-buffered saline Room temperature Small interfering RNA Tris-buffered saline with Tween-20 Tetratricopeptide Transport protein particle TRAPP complex Introduction To date, more than 50 different high-throughput studies aimed at cataloging the constituents of cilia and centrosomes from various species have been published.
Cilia: Methods and Protocols by Peter Satir, Søren Tvorup Christensen