Journal: Journal of visualized experiments : JoVE

Article Title: Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

doi: 10.3791/60824

Figure Lengend Snippet: Materials

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Generation of clonal bacterial colonies carrying the shRNA plasmids list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Obtain glycerol stocks of E. coli carrying target-specific shRNA plasmids and a control plasmid from commercial sources ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Name Company Catalog Number Comments Acetone Fisher Chemical 191784 Agar Fisher Bioreagents BP1423 Ampicillin Fisher Bioreagents BP1760–5 Automated cell counter Countesse II Invitrogen A27977 Bradford Reagent Thermo Scientific P4205987 C2C12 cells ATCC CRL-1772 Chamber slides Invitrogen C10283 Chloroform Fisher Chemical 183172 DMEM GIBCO 11965–092 DMSO Fisher Bioreagents BP231–100 DNase I (Amplification Grade) Invitrogen 18068015 Fetal bovine serum VWR 97068–085 GAPDH EMD Millipore MAB374 Glycine VWR Life Sciences 19C2656013 Goat-anti-mouse secondary antibody (IRDYE 800CW) Li-Cor C90130–02 Goat-anti mouse secondary antibody (Rhodamine-red) Jackson Immune Research 133389 HCl Fisher Chemical A144S Incubator (Shaker) Denville Scientific Corporation 1704N205BC105 Mercaptoethanol Amresco, VWR Life Sciences 2707C122 Midiprep plasmid extraction kit Qiagen 12643 Myosin 4 (myosin heavy chain) Invitrogen 14–6503–82 Mounting medium Invitrogen 2086310 NaCl VWR Life Sciences 241 non-ionic surfactant/detergent VWR Life Sciences 18D1856500 Paraformaldehyde MP 199983 PBS Fisher Bioreagents BP399–4 PEI Polysciences 23966–1 Penicillin/streptomycin antibiotics GIBCO 15140–122 Petridishes Corning 353003 Polypropylene tubes Fisherbrand 149569C Protease inhibitor cocktail tablets Roche 33576300 Puromycin Fisher Scientific BP2956100 PCR (Real Time) Applied Biosystems 4359284 Reaction tubes Eppendorf 22364111 Reverse Transcription Master Mix Applied Biosystems 4368814 RIPA buffer Thermo Scientific TK274910 sh control plasmid Sigma-Aldrich 07201820MN sh 3086 plasmid Sigma-Aldrich TRCN0000092578 sh 972 plasmid Sigma-Aldrich TRCN0000092579 sh 1977 plasmid Sigma-Aldrich TRCN0000092582 Spectrophotometer (Nanodrop) Thermo Scientific NanoDrop One C SYBR Green Reagent Master Mix Applied Biosystems 743566 Trichloroacetic acid Acros Organics 30145369 Trizol reagent Ambion 254707 Trypan blue GIBCO 15250–061 Tryptone Fisher Bioreagents BP1421 Trypsin EDTA 0.25% Gibco-Life Technology Corporation 2085459 Water (DEPC treated and nuclease free) Fisher Bioreagents 186163 Western blotting apparatus Biorad Mini Protean Tetra Cell Yeast extract Fisher Bioreagents BP1422 Open in a separate window Materials NOTE: Three different shRNA plasmids were used, targeting different regions of the murine Adamtsl2 mRNA.

Techniques: Amplification, Plasmid Preparation, Protease Inhibitor, Spectrophotometry, SYBR Green Assay, Western Blot