By Hans-Joachim Anders, Adriana Migliorini
Innate DNA and RNA popularity: process and Protocols provides demonstrated experimental thoughts to dissect nucleic acid sensing in-vitro and in-vivo resources. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols and tips about troubleshooting and heading off identified pitfalls.
Authoritative and practical, Innate DNA and RNA popularity: procedure and Protocols offers a source for immunologists, molecular biologists, virologists, microbiologists and researchers learning how the innate immune process handles nucleic acids from endogenous or overseas sources.
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Extra info for Innate DNA and RNA Recognition: Methods and Protocols
Curr Opin Immunol 20(4):389–395. 007 Poeck H, Besch R, Maihoefer C, Renn M, Tormo D, Morskaya SS, Kirschnek S, Gaffal E, Landsberg J, Hellmuth J, Schmidt A, Anz D, Bscheider M, Schwerd T, Berking C, Bourquin C, Kalinke U, Kremmer E, Kato H, Akira S, Meyers R, Hacker G, Neuenhahn M, Busch D, Ruland J, Rothenfusser S, Prinz M, Hornung V, Endres S, Tuting T, Hartmann G (2008) 5′-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat Med 14(11):1256–1263. 1887 Kubler K, Gehrke N, Riemann S, Bohnert V, Zillinger T, Hartmann E, Polcher M, Rudlowski C, Kuhn W, Hartmann G, Barchet W (2010) Targeted activation of RNA helicase retinoic acid-inducible gene-I induces proimmunogenic apoptosis of human ovarian cancer cells.
Transform competent Rosetta (DE3) E. coli with pET vector expressing 6His-MBP-TEV-RIG-I 1-797 and spread to LB agar plate containing 30 μg chloramphenicol, 100 μg/ml ampicillin. Incubate the plates overnight at 37 °C. 2. Remove the plate from the incubator in the morning and store at room temperature until late afternoon. 3. Pick up 4–5 colonies and inoculate a starter culture of 10 ml in LB containing 30 μg chloramphenicol, 100 μg/ml ampicillin per a liter of induction culture. Incubate overnight at 37 °C with 220 rpm) shaking.
Shake off the rest of the cells until all cells have become detached (see Note 2). Wash flasks with 10 ml of ice-cold PBS (see Note 3). Collect cells, media and PBS in 50 ml tubes and put them on ice. 5. Centrifuge cells at 400 × g for 7 min at 4 °C and discard the supernatant. Resuspend the cell pellet in 1 ml of PBS and pool cells from different tubes of one condition and add 30 ml cold PBS. Centrifuge again and repeat this washing procedure three times. 6. After washing resuspend cells in 1 ml of lysis buffer (see Note 4).
Innate DNA and RNA Recognition: Methods and Protocols by Hans-Joachim Anders, Adriana Migliorini