Download RNA Scaffolds: Methods and Protocols by Luc Ponchon PDF

By Luc Ponchon

This quantity presents ideas on contemporary advancements that use RNA scaffolds as molecular instruments. RNA Scaffolds: tools and Protocols courses readers via equipment on a number of domain names as molecular biology, nanotechnologies, and structural biology. Written for the Methods in Molecular Biology sequence, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols and tips about troubleshooting and fending off identified pitfalls.

Practical and authoritative, RNA Scaffolds: tools and Protocols will function a useful reference for these attracted to extra examine into this interesting field.

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Additional info for RNA Scaffolds: Methods and Protocols

Sample text

12. Analyze the fractions by electrophoresis on 11 % SDSPAGE. Following electrophoresis, stain the gel with staining solution and destain with destaining solution. Place the gel on shrink wrap (see Note 5) and place on a white paper. Reveal the RNA by UV shadowing with the handheld UV lamp placed above the gel. The protein appears as a blue band and RNA and grey band (Fig. 2). Expression and Purification of RNA–Protein Complexes in Escherichia coli 29 Fig. 2 Co-expression of AtRNA-mala/His6-MS2 coat protein in XL1-Blue E.

Coli BL21 (DE3), stored at −80 °C (Novagen) (see Note 2). 3. Ampicillin stock solution: 100 mg/mL. 22 µm filter-sterilize. 5 mL vials and store at −20 °C. 4. Luria Broth (LB medium) [10]: weigh out 10 g of tryptone, 5 g of yeast extract powder, and 10 g of sodium chloride. 0. Sterilize by autoclaving. 5. 75 g of agar to 250 mL of LB medium before autoclaving. After autoclaving, allow the medium to cool to approximately 55 °C and add 125 µL of ampicillin stock solution. Mix by gentle swirling. Then pour the plates of 10–15 mL in a sterile disposable Petri dish (Ø 9 cm); use directly or store at 4 °C for up to 2 weeks.

And 37 °C. 3. Add 25 mL of the saturated overnight culture to 1 L fresh 2xTY medium containing 100 µg/mL ampicillin and 20 µg/ mL chloramphenicol in a baffle-bottomed shake flask. 4. m. 6. 5. Collect a sample for protein production analysis (see Note 3). 6. Add IPTG to a final concentration of 1 mM. Continue shaking for 3–4 h. 7. Collect a second sample for protein production analysis (see Note 3). 8. Pellet 5 mL of culture by centrifugation for 10 min at 4,000 × g and 4 °C for RNA production analysis.

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