By Tina B. Miranda, Ty C. Voss, Gordon L. Hager (auth.), Yaron Shav-Tal (eds.)
As imaging applied sciences and techniques have developed, the scope of sure imaging suggestions has moved a long way past the creation of in basic terms illustrative pictures or beautiful time-lapse videos to supplying the scientist with a wealthy diversity of how to degree and quantify the organic technique and consequence of gene expression. In Imaging Gene Expression: tools and Protocols, specialist authors supply up to date ways and protocols that scientists within the box have constructed, which might profit the wider medical group. Divided in 3 handy components, this distinct ebook covers the output of a gene, specifically the RNA molecules which are transcribed from the gene and how in which those molecules might be tracked or quantified in mounted or dwelling cells, protocols that target the gene, DNA, or chromatin, in addition to numerous methods through which nuclear strategies intertwined with gene expression might be and quantified in residing cells in addition to techniques for learning numerous sub-nuclear buildings present in eukaryotic cells. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective topics, lists of fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and up to date, Imaging Gene Expression: equipment and Protocols will serve researchers operating towards imaging within the context of whole organisms.
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Exposure time was 20 ms for the green channel (488 nm) and 10 ms for the red channel (561 nm). 1 % and the EMCCD camera gain to 300. 5 Assessing the Cell State After Long-Term Live-Cell Imaging The color of the culture medium and cell morphology should always be recorded at the beginning and end of any live-cell imaging experiment. Useful indicators of possible phototoxicity include cell morphology, cell cycle, and cell division perturbations. In the ESC line we use as a model system, the nucleoli have a different background staining than the rest of the nucleus.
5. Change medium and trypsinize the transfected cells the following morning and plate out 10 % and 90 % of the cells onto two pre-gelatinized 10-cm tissue culture dishes and culture the cells in ES medium for another day. 6. , hygromycin B; see Note 1). 7. After selection begins, medium should be changed every day until clone picking. Nonresistant cells should die during the next 1–5 days depending on the drug used. 2 ESC Clone Picking 1. Discrete ESC colonies should become visible by eye after approximately 7–10 days of culture.
18. DNA counterstaining solution: 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in 2× SSC. 19. 0 in PBS. 20. Eppendorf Centrifuge 5417R. 21. Eppendorf Concentrator plus. 22. Eppendorf Thermomixer comfort. 23. Liquiport Liquid pump. 24. Shake’N’Bake Hybridization Oven. 4 Plasmids 1. pBROAD3-TetR-ICP22-mCherry. 2. pBROAD3-TetR-ICP22-EGFP. 3. pBROAD3-mCherry-PCNA. 5 ES Cell Lines 1. PGK 31′-60 (PGK 2TetO TetR-mCherry). 2. PGK 31′-T12 TetR-EGFP). TetR-ICP22-EGFP 12C (PGK 2TetO 18 3 Tim Pollex et al.