Mutagenesis:Article Title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation
Article Snippet: .. Unnatural-Amino-Acid Mutagenesis of FLAG-αNTD I -GSGGSG-αNTD II , β, β' or ω list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding FLAG-αNTD I -GSGGSG-αNTD II , β, β’, or ω ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol, or 50 μg/mL chloramphenicol and 40 μg/mL kanamycin, or 50 μg/mL chloramphenicol and 100 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) 50 mg/mL Chloramphenicol (filter-sterilized) 40 mg/mL Kanamycin (filter-sterilized) 1000 mg/mL Ampicillin (filter-sterilized) 20% Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) Buffer D (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM DTT) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl2, and 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 50 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker 2.3.2. .. Unnatural-Amino-Acid Mutagenesis of σ 70 list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker 2.4.
Article Title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation
Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding FLAG-αNTD I -GSGGSG-αNTD II , β, β’, or ω ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol, or 50 μg/mL chloramphenicol and 40 μg/mL kanamycin, or 50 μg/mL chloramphenicol and 100 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) 50 mg/mL Chloramphenicol (filter-sterilized) 40 mg/mL Kanamycin (filter-sterilized) 1000 mg/mL Ampicillin (filter-sterilized) 20% Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) Buffer D (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM DTT) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl2, and 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 50 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker
Plasmid Preparation:Article Title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation
Article Snippet: .. Unnatural-Amino-Acid Mutagenesis of FLAG-αNTD I -GSGGSG-αNTD II , β, β' or ω list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding FLAG-αNTD I -GSGGSG-αNTD II , β, β’, or ω ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol, or 50 μg/mL chloramphenicol and 40 μg/mL kanamycin, or 50 μg/mL chloramphenicol and 100 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) 50 mg/mL Chloramphenicol (filter-sterilized) 40 mg/mL Kanamycin (filter-sterilized) 1000 mg/mL Ampicillin (filter-sterilized) 20% Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) Buffer D (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM DTT) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl2, and 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 50 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker 2.3.2. .. Unnatural-Amino-Acid Mutagenesis of σ 70 list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker 2.4.
Article Title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation
Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding FLAG-αNTD I -GSGGSG-αNTD II , β, β’, or ω ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol, or 50 μg/mL chloramphenicol and 40 μg/mL kanamycin, or 50 μg/mL chloramphenicol and 100 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) 50 mg/mL Chloramphenicol (filter-sterilized) 40 mg/mL Kanamycin (filter-sterilized) 1000 mg/mL Ampicillin (filter-sterilized) 20% Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) Buffer D (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM DTT) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl2, and 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 50 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker
Sterility:Article Title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation
Article Snippet: .. Unnatural-Amino-Acid Mutagenesis of FLAG-αNTD I -GSGGSG-αNTD II , β, β' or ω list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding FLAG-αNTD I -GSGGSG-αNTD II , β, β’, or ω ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol, or 50 μg/mL chloramphenicol and 40 μg/mL kanamycin, or 50 μg/mL chloramphenicol and 100 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) 50 mg/mL Chloramphenicol (filter-sterilized) 40 mg/mL Kanamycin (filter-sterilized) 1000 mg/mL Ampicillin (filter-sterilized) 20% Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) Buffer D (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM DTT) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl2, and 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 50 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker 2.3.2. .. Unnatural-Amino-Acid Mutagenesis of σ 70 list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker 2.4.
Article Title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation
Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding FLAG-αNTD I -GSGGSG-αNTD II , β, β’, or ω ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol, or 50 μg/mL chloramphenicol and 40 μg/mL kanamycin, or 50 μg/mL chloramphenicol and 100 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) 50 mg/mL Chloramphenicol (filter-sterilized) 40 mg/mL Kanamycin (filter-sterilized) 1000 mg/mL Ampicillin (filter-sterilized) 20% Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) Buffer D (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM DTT) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl2, and 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 50 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plasmid encoding σ 70 ( ) Plasmid pEVOL-pAzF ( 29 ; provided by P. Schultz, The Scripps Research Institute, La Jolla CA) Chemically competent E. coli strain BL21(DE3) (Invitrogen) QuikChange II Site-Directed Mutagenesis Kit (Agilent) LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl; autoclave-sterilized) TYE agar containing 50 μg/mL chloramphenicol or containing 35 μg/mL chloramphenicol and 200 μg/mL ampicillin (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar; autoclave-sterilized without antibiotics; supplemented with antibiotic after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates; ~25 mL/plate) M9+ medium (1 × M9 minimal salts, 0.4% D-glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1 mM FeSO 4 , 3 nM (NH 4 ) 6 Mo 7 O 24 , 400 nM H 3 BO 3 , 30 nM CoCl 2 , 10 nM CuSO 4 , 80 nM MnCl 2 , 20 nM ZnSO 4 , 0.4 mg/L choline chloride, 0.5 mg/L folic acid, 0.5 mg/L nicotinamide, 1 mg/L myo-inositol, 1 mg/L pyridoxal HCl, 2 mg/L thiamine HCl, 0.05 mg/L riboflavin, 1 mg/L biotin; prepared freshly before use from 10× M9 minimal salts, 20% glucose, 1 M MgSO 4 , 1 M CaCl 2 , 0.2 M FeSO 4 , 1000× vitamins, and autoclaved water) 50 mg/mL Chloramphenicol (filter-sterilized) 100 mg/mL Ampicillin (filter-sterilized) 20% L-Arabinose (filter-sterilized) 1 M IPTG (filter-sterilized) (Gold Biotechnology) 4-Azido-L-phenylalanine (ChemImpex) 10× M9 minimal salts (67.8 g/L Na 2 HPO 4 -7H 2 O, 30 g/L KH 2 PO 4 , 10 g/L NH 4 Cl, 5 g/L NaCl; autoclave-sterilized) 20% D-Glucose (prepared in autoclaved water, filter-sterilized) 1 M MgSO 4 (prepared in autoclaved water, filter-sterilized) 1 M CaCl 2 (prepared in autoclaved water, filter-sterilized) 0.2 M FeSO 4 1000× Vitamins (0.4 g/L choline chloride, 0.5 g/L folic acid, 0.5 g/L nicotinamide, 1g/L myo-inositol, 1 g/L pyridoxal HCl, 2 g thiamine HCl, 0.05 g/L riboflavin, and 1 g/L biotin) (Sigma-Aldrich) Buffer H (10 mM potassium acetate, pH 7.0, 10 mM KCl, 50 mM CaCl 2 , and 10% glycerol) Buffer I (40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 10% glycerol) 2.8 L Triple-baffled Fernbach flask (autoclave-sterilized) (Bellco Glass) 250 mL Erlenmeyer flask (VWR) 1.7 mL Polypropylene microcentrifuge tubes (Denville Scientific) 14 mL Polypropylene culture tubes with snap closures (autoclave-sterilized) (VWR) Microcentrifuge (e.g., Eppendorf 5417C) Platform shaker
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