By Xiuzhong Zheng, Anastasiya Epstein, Hannah L. Klein (auth.), Hideo Tsubouchi (eds.)
Homologous recombination is critical in quite a few facets of DNA metabolism, together with harm fix, replication, telomere upkeep, and meiosis, and yeast genetics has effectively supplied a framework for the mechanism of homologous recombination. Divided into 4 handy sections, DNA Recombination: equipment and Protocols covers contemporary recommendations that most sensible make the most of some great benefits of the yeast approach, prescribing to the idea that yeast will hold serving as a superb version organism to review homologous recombination. Chapters have additionally been incorporated for such exceptions because the team of genes interested by recombination which are stumbled on exclusively in larger eukaryotes, corresponding to BRCA2. and looking out ahead, an important step towards realizing the homologous recombination procedure is to isolate the computing device and enable it paintings in a try tube. figuring out the layout by way of learning the looks and behaviour of the equipment as a unmarried molecule can be a tremendous milestone towards knowing the mechanism of motion of the equipment. recommendations overlaying those issues have additionally been integrated. Written within the winning Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible protocols, and notes on troubleshooting and warding off identified pitfalls.
Authoritative and simply available, DNA Recombination: tools and Protocols serves as a terrific consultant to scientists of all backgrounds with its well-honed methodologies and strives to carry the reader to the subsequent point of figuring out relating to this very important subject.
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Additional resources for DNA Recombination: Methods and Protocols
6. Further time-point samples are taken as appropriate (see Note 15). 5- or 2-ml tubes, pelleted, and stored at –80◦ C. 3. Preparation of Agarose–Cell Plugs 1. Agarose solution is prepared and placed in a beaker of water, microwaved carefully until all agarose is dissolved, and is then kept at 50◦ C in a hotblock. 2. The base of the plug molds is sealed with tape and zymolyase solution is prepared and kept on ice. 3. Cell pellets are thawed on ice, resuspended in 66 μl (per 10 ml original sample) zymolyase solution, and allowed to come to room temperature.
Curr Biol 14, 2107–2112. 25. A. A. (2008) Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae. PLoS Genet 4, e1000264. Chapter 3 Characterization of Meiotic Recombination Initiation Sites Using Pulsed-Field Gel Electrophoresis Sarah Farmer, Wing-Kit Leung, and Hideo Tsubouchi Abstract High levels of homologous recombination are induced during meiosis. This meiotic recombination is initiated by programmed formation of DNA double-strand breaks (DSBs) by a conserved meiosis-specific protein, Spo11.
2. 25 N HCl to the tray containing the CHEF gel and gently shake for1 45–60 min. 3. Rinse gel briefly with water; treat with the alkaline solution for 30–60 min. 4. Neutralize with neutralization buffer for 30 min. 5. Cut a Hybond N+ membrane, wet first in water, and then soak for 10–15 min in 10× SSC. 6. Select a suitable method for Southern transfer. For example, use capillary method or a vacuum blotter according to the manufacturer’s instructions. 7. Rinse the membrane with 10× SSC. Dry the membrane or UV-crosslink with Stratalinker (120 mJ/cm2 ).