Chromosome Architecture: Methods and Protocols by Mark C. Leake

By Mark C. Leake

This quantity information a invaluable selection of protocols and experiences, akin to rising experimental and theoretical ways. those ways have led to a considerable development within the figuring out of chromosome architecture. Chromosome structure: tools and Protocols publications readers via state of the art interdisciplinary tools which enable for an knowing of structure of chromosomes with particularly better answer, either by way of area and time. Written within the hugely winning Methods in Molecular Biology series layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and pointers on troubleshooting and keeping off recognized pitfalls.

Authoritative and cutting-edge, Chromosome structure: tools and Protocols aims to make sure winning ends up in the extra research of this important field.

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44 Anjana Badrinarayanan and Mark C. Agarose pads can dry out or dessicate when kept for a long period during imaging (especially at high temperatures such as 37 °C). In order to prevent this, gene frames are used. As stated earlier, M9–glycerol provides ideal growth conditions for microscopy-based experiments in E. coli. Another important advantage of using M9–glycerol over LB is the lower autofluorescence in M9. Background fluorescence can pose a problem during imaging and in particular, during analysis of fluorescence intensity in the cell.

5 Single-Molecule Imaging Instrumentation We perform single-molecule using a custom-built microscope, based on an Olympus IX81 base (Fig. 2). The system is set-up to allow epifluorescence, TIRF or inclined illumination of the sample. Inclined illumination schemes (including HILO) can be used to improve the S/N ratio of the imaging, by reducing the illumination of molecules above and below the focal plane of interest. TIRF illumination cannot be employed since the NPCs are typically located above the shallow (~100 nm) evanescent field of illumination.

In the picture, you will be able to see typically two distinct MukB-YFP foci per cell. The aim of the experiment is to bleach one of the two foci and record fluorescence recovery after bleaching (see Note 8 on use of cephalexin to elongate cells). Open the FRAP module to set up bleaching conditions (see Note 9 on FRAP calibration). Pulse bleach is ideally done with 6–15 % laser intensity for 15 ms. The number of cycles of bleaching is limited to 1. A region of interest (ROI) is drawn around the focus to be bleached.

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