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By Ivo L. Hofacker, Ronny Lorenz (auth.), Christina Waldsich (eds.)
Focusing on in vitro and intracellular RNA constitution formation, RNA Folding: equipment and Protocols offers a accomplished selection of experimental protocols that are appropriate to dissect RNA folding pathways and to symbolize the constitution of RNA folding intermediates at nucleotide or perhaps atomic answer. The awarded concepts contain robust instruments with a protracted culture in RNA study in addition to extra complicated, novel equipment, therefore the tools span a number of disciplines, together with molecular biology, biochemistry, biophysics, and computational biology. Written within the hugely winning Methods in Molecular Biology sequence layout, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, easily reproducible laboratory protocols, and tips about troubleshooting and fending off identified pitfalls.
Practical and authoritative, RNA Folding: tools and Protocols serves as an important reference for researchers trying to achieve insights into the secrets and techniques of this staggering macromolecule.
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Extra resources for RNA Folding: Methods and Protocols
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Purify the complementary DNA oligos that will form the dsDNA templates used for in vitro transcription of the different ribozymes’ cores by 10 % denaturing PAGE. 34 Mélanie Meyer and Benoît Masquida 3. Mix 20 μg of the forward and the reverse pure template DNA oligos and add KCl up to a final concentration of 500 mM. To hybridize the template DNA oligos heat the sample for 1 min at 95 °C and allow it to cool to room temperature for 10 min. 4. 8 μL of alkaline phosphatase (1 U/μL) (see Note 4), T7 RNA polymerase as indicated by your provider (see Note 5), water to 200 μL.
Importantly, this approach is especially suitable for short RNAs because it does not require appending flanking nucleotides for annealing cDNA primer or for eliminating noise near the RNA 5′ end, both of which are required for SHAPE analysis of small RNAs [9]. The enzymatic digestion method is based on the sequence and structural preferences of natural nucleases (nuclease specificities and reactivities are reviewed in [10]). In brief, the method is to allow the RNA to fold into its native conformation, digest briefly with nuclease, and then identify the cleavage sites by electrophoretic separation of the cleavage fragments.
6. N,N,N′,N′-tetramethylethylene-1,2-diamine (TEMED). Store at 4 °C. 7. 89 M boric acid, 20 mM EDTA. Store at room temperature. 8. Vertical electrophoresis equipment (medium and sequencing sizes). RNA Structural Analysis by Enzymatic Digestion 45 9. Saran wrap. 10. X-ray film and film developer, and/or phosphorimager and screen. 11. Fluorescent stickers if available. 12. Light box if available. 13. Clean razor blade and tweezers. 14. 03 mm pore size). 15. 0. Store at room temperature. 16. Rotator.