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This quantity comprises entire and up to date assurance of all nucleic-acid-specific steps utilized in X-ray crystallography, from macromolecule creation to constitution choice. Chapters devoted to RNA training and crystallogenesis may be of curiosity to newcomers, whereas chapters enthusiastic about facts assortment, phasing and refinement could be really valuable to researchers with a better point of craftsmanship. numerous useful case stories also are awarded within the final a part of the ebook. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and tips about troubleshooting and averting identified pitfalls.
Authoritative and thorough, Nucleic Acid Crystallography: tools and Protocols provides protocols which are aimed toward either researchers and scholars who're attracted to the structural biology of DNA or RNA, by myself or in advanced with proteins or ligands.
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Serganov A, Patel DJ (2012) Metabolite recognition principles and molecular mechanisms underlying riboswitch function. Annu Rev Biophys 41:343–370 10. Milligan JF, Groebe DR, Witherell GW et al (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798 11. Pikovskaya O, Serganov AA, Polonskaia A et al (2009) Preparation and crystallization of riboswitch-ligand complexes. Methods Mol Biol 540:115–128 12. Peselis A, Serganov A (2012) Structural insights into ligand binding and gene expression control by an adenosylcobalamin riboswitch.
1, step 7. 2. Run the test reactions for 3–4 h at 37 °C. The appearance of a white precipitate due to magnesium pyrophosphate is most of the time visible and can be used to monitor the transcription efficiency. 3. Store at −20 °C or analyze immediately by SDS-PAGE. 0. 46 Clément Dégut et al. 4. To analyze the products of transcription, prepare a SDS-PAGE gel using the same materials as those used for protein SDSPAGE analysis. , a 14 % gel for a 92 nucleotides tRNA. 8, 50 μL SDS 20 % and adjust the volume to 10 mL with water.
6. 15 μL volumes of the RNA and crystallization solutions into the wells of the plate. Seal the plate with Clear Seal film. Store the plate at the desired temperature. 7. Allow the drops in the plates to equilibrate for at least 1 day before checking them with a stereomicroscope at a magnification range of ~5–10×. A polarizing filter is highly recommended for locating small crystals (Fig. 3a, b, g, h). 8. Once conditions that produce crystals have been determined, reproduce crystals in 2 μL hanging drops.