bl21  (New England Biolabs)


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    Name:
    BL21 Competent E coli
    Description:
    BL21 Competent E coli 20x0 05 ml
    Catalog Number:
    c2530h
    Price:
    204
    Size:
    1 ml
    Category:
    Competent Bacteria
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    New England Biolabs bl21
    BL21 Competent E coli
    BL21 Competent E coli 20x0 05 ml
    https://www.bioz.com/result/bl21/product/New England Biolabs
    Average 95 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    bl21 - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor
    Article Snippet: Strains, plasmids and media Escherichia coli (E. coli ) DH5α was used for routine cloning and plasmid propagation (NEB, #C2987I). .. E. coli BL21 strain (NEB, #C2530H) was used as production strain harboring the original pathway eAKP672.

    Article Title: RAB21 Activity Assay Using GST-fused APPL1
    Article Snippet: .. 50 ml conical tube (Sarstedt AG & CO, catalog number: 62.547.004) 100 mm Tissue Culture Dish (Corning, catalog number: 430167) 0.22 μm filtering unit (Genesee Scientific Corporation, catalog number: 25-227) Cell lifter (Corning, catalog number: 3008) Escherichia coli BL21 (New England Biolabs, catalog number: C2530H) pGEX-5X-3 (GE Healthcare, catalog number: 27-4586-01) pGEX5X3-Happl1 (aa 5-419) (self made) ( ) pAcEGFP-C1 vector (Clontech, catalog number: 632470) pEGFP-C1:RAB21 wild type [human RAB21 cloned in pAcEGFP-C1 (this construct was used to generate the stable HeLa M cell line)] (self made) ( ) EGFP:RAB21wt (wildtype) stably-transfected HeLa M cells (self made) ( ) Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Biopioneer, catalog number: c0012) Ampicillin Sodium Salt (Crystalline Powder) (Thermo Fisher Scientific, catalog number: ) BD Bacto™ Tryptone (Thermo Fisher Scientific, catalog number: DF0123173) Yeast Extract (Thermo Fisher Scientific, catalog number: 212750) Glutathione Sepharose 4B beads (GE Healthcare, catalog number: 17-0756-01) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-5) Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml) Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15140-122”. .. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200-056) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25200-056”.

    Amplification:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: G-blocks were PCR amplified using Forward primer 5′-TTATTCGAATTCGCGGCCGCTTCTAGAG and Reverse primer 5′-GGATTTCTGCAGCGGCCGCTACTAGTA with the following conditions: in a 50 μL reaction: 10 μL 5x HF buffer (New England Biolabs, NEB #E0553L), 2 μL 10 mM dNTP mix (NEB #N0447L), 1 μL 100 μM forward and reverse primer, 25 ng g block template, and 0.5 μL phusion polymerase. .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: The coding sequence of CsLOB1 of 714 bp was amplified with primers containing Eco RI/ Sal I restriction sites, and subcloned into the protein expression vector pGEX‐4T‐1, named pGEX‐4T‐1‐CsLOB1, using Q5® High‐Fidelity 2× Master Mix (New England Biolabs, Ipswich, MA, USA). .. The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA).

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Constructs containing the complete RNF114 sequence were amplified using primers containing 20 base pair homology to a pET24a plasmid (Novagen) that also contained a His8 -MBP-TEV sequence between Nde1 and BamH1 restriction sites. .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    Filtration:

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Purified proteins were desalted by gel filtration with Zeba spin columns (7000 MWCO, Thermo Fischer Scientific, USA) and subsequently labeled with ten-fold molar excess of Alexa647-NHS (Thermo Fischer Scientific, USA) in 0.1 mM KH2 PO4 buffer at RT overnight.

    Stable Transfection:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: Netrin-1 protein was either obtained from R & D (Minneapolis, MN, USA) or purified with anti-Myc tag affinity matrix from the conditioned media of HEK cells stably secreting netrin-1. .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA).

    Article Title: RAB21 Activity Assay Using GST-fused APPL1
    Article Snippet: .. 50 ml conical tube (Sarstedt AG & CO, catalog number: 62.547.004) 100 mm Tissue Culture Dish (Corning, catalog number: 430167) 0.22 μm filtering unit (Genesee Scientific Corporation, catalog number: 25-227) Cell lifter (Corning, catalog number: 3008) Escherichia coli BL21 (New England Biolabs, catalog number: C2530H) pGEX-5X-3 (GE Healthcare, catalog number: 27-4586-01) pGEX5X3-Happl1 (aa 5-419) (self made) ( ) pAcEGFP-C1 vector (Clontech, catalog number: 632470) pEGFP-C1:RAB21 wild type [human RAB21 cloned in pAcEGFP-C1 (this construct was used to generate the stable HeLa M cell line)] (self made) ( ) EGFP:RAB21wt (wildtype) stably-transfected HeLa M cells (self made) ( ) Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Biopioneer, catalog number: c0012) Ampicillin Sodium Salt (Crystalline Powder) (Thermo Fisher Scientific, catalog number: ) BD Bacto™ Tryptone (Thermo Fisher Scientific, catalog number: DF0123173) Yeast Extract (Thermo Fisher Scientific, catalog number: 212750) Glutathione Sepharose 4B beads (GE Healthcare, catalog number: 17-0756-01) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-5) Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml) Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15140-122”. .. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200-056) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25200-056”.

    Synthesized:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: Gene cassettes were designed in CLC Main (Qiagen) and synthesized as g-blocks by IDT. .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: For the second method, DNA encoding the complete human isoform of RNF114 (Uniprot id: Q9Y508) was codon optimized for expression in E. coli and synthesized by Integrated DNA Technologies. .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    Construct:

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H). .. The following day, a single transformed colony was used to inoculate 50 mL of nutrient rich LB medium containing kanamycin (50 μg/mL) and was incubated at 37 °C overnight, with agitation (250 rpm).

    Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin
    Article Snippet: GST Pull-Down Assays The GST-SMAD4 fusion protein was induced with 0.1 mM IPTG in BL21 (C2530H) Escherichia coli competent cells (New England BioLabs, NEB, MA, United States) ) transformed with pGEX-2TK-SMAD4 and after 4 h, the bacteria were lysed in 2% sarkosyl-STE buffer (10 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) and sonicated. .. HEK293-I3A cells were transfected with pCMV-HA-MyD88 or one of the three pCMV-HA-MyD88 deletion constructs.

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: We have previously shown that these two katanin constructs have similar high severing activity (Bailey et al., ). .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon.

    Article Title: Proteome-wide enrichment of proteins modified by lysine methylation
    Article Snippet: Plasmids: 3×MBT-pGEX6P-1 and 3×MBTD355N -pGEX6P-1 constructs (AddGene plasmid nos. .. 46987 and 46988, respectively) Mammalian SILAC medium: DMEM (Thermo Scientific/Pierce, cat. no. 88420) and RPMI (Thermo Scientific/Pierce, cat. no. 88421) Dialyzed FBS for SILAC (Thermo Scientific/Pierce, cat. no. 88440) Yeast SILAC medium (based on Kubota et al. ): Dropout mix minus leucine, lysine, arginine and adenine (US Biological, cat. no. D9515F) Yeast nitrogen base without amino acids (Sigma, cat. no. Y0626) Leucine (Sigma, cat. no. L8912) Adenine (Sigma, cat. no. A8626) Glucose (Invitrogen-Gibco, cat. no. 15023021) SILAC amino acids: L-lysine-2HCl (Thermo Scientific/Pierce, cat. no. 88429), L-arginine-HCl (Thermo Scientific/Pierce, cat. no. 88427), 2 H4 -L-lysine-2HCl (Thermo Scientific/Pierce, cat. no. 88438), 13 C6 -L-arginine-HCl (Thermo Scientific/Pierce, cat. no. 88433), 15 N2 13 C6 -L-lysine-2HCl (Thermo Scientific/Pierce, cat. no. 88209), 15 N4 13 C6 -L-arginine-HCl (Thermo Scientific/Pierce, cat. no. 89990), L-proline (Thermo Scientific/Pierce, cat. no. 88430) Yeast strain from any background (for example, Fisher cat. no. NC0537738), but they must be auxotrophic for arginine and lysine ( arg4 lys2 ) as described in ref. LB medium (Sigma, cat. no. L3522) BL21-competent Escherichia coli (New England BioLabs, cat. no. C2530 H) Ampicillin sodium salt (Sigma, cat. no. A0166) IPTG (Sigma, cat. no. I5502) Tris base (Fisher, cat. no. BP152) Hydrochloric acid (HCl; Fisher, cat. no. A144) !

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: We will refer to the Xenopus construct as Xl-p60 and the human construct as GFP-Hu-p60. .. The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA).

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: For TUBB3 RNAi rescue experiments, an RNAi-resistant construct was created by introducing seven silent point mutations in the target sequences. .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA).

    Article Title: RAB21 Activity Assay Using GST-fused APPL1
    Article Snippet: .. 50 ml conical tube (Sarstedt AG & CO, catalog number: 62.547.004) 100 mm Tissue Culture Dish (Corning, catalog number: 430167) 0.22 μm filtering unit (Genesee Scientific Corporation, catalog number: 25-227) Cell lifter (Corning, catalog number: 3008) Escherichia coli BL21 (New England Biolabs, catalog number: C2530H) pGEX-5X-3 (GE Healthcare, catalog number: 27-4586-01) pGEX5X3-Happl1 (aa 5-419) (self made) ( ) pAcEGFP-C1 vector (Clontech, catalog number: 632470) pEGFP-C1:RAB21 wild type [human RAB21 cloned in pAcEGFP-C1 (this construct was used to generate the stable HeLa M cell line)] (self made) ( ) EGFP:RAB21wt (wildtype) stably-transfected HeLa M cells (self made) ( ) Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Biopioneer, catalog number: c0012) Ampicillin Sodium Salt (Crystalline Powder) (Thermo Fisher Scientific, catalog number: ) BD Bacto™ Tryptone (Thermo Fisher Scientific, catalog number: DF0123173) Yeast Extract (Thermo Fisher Scientific, catalog number: 212750) Glutathione Sepharose 4B beads (GE Healthcare, catalog number: 17-0756-01) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-5) Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml) Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15140-122”. .. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200-056) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25200-056”.

    Incubation:

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. Escherichia coli BL21 was cultured in 10 mL of Luria–Bertani (LB) medium at 37 °C overnight; 0.1 mL of the overnight culture was transferred into 50 mL of LB medium containing 50 µ m kanamycin and incubated at 37 °C until A 600 nm reached OD = 0.6.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H). .. The following day, a single transformed colony was used to inoculate 50 mL of nutrient rich LB medium containing kanamycin (50 μg/mL) and was incubated at 37 °C overnight, with agitation (250 rpm).

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: Double-stranded (ds) RNAs for intererence in S2 cells have been generated using the following primers: T3-Snap29 5′-TAATACGACTCACTATAGGGAGA AACCCAGGAGGTGGGTAAG-3′ T7- Snap29 5′-AATTAACCCTCACTAAAGGGAGA ATGTTATCCAGCAATTCATTTTG-3′ DsRNA were in vitro transcribed with the T3 (Promega, P208C) and T7 (Promega, P207B) polymerase according to manufacturer's instructions, annealed, and incubated with the cells at a final concentration of 15ug/106 cells for 72 h. Multiple sequence analysis was performed using ClustalX using standard parameters. .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin
    Article Snippet: GST Pull-Down Assays The GST-SMAD4 fusion protein was induced with 0.1 mM IPTG in BL21 (C2530H) Escherichia coli competent cells (New England BioLabs, NEB, MA, United States) ) transformed with pGEX-2TK-SMAD4 and after 4 h, the bacteria were lysed in 2% sarkosyl-STE buffer (10 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) and sonicated. .. Ten micrograms of GST-SMAD4 fusion protein or GST (as control) were incubated with Glutathione Sepharose 4B (GE Healthcare) for 1 h at 4°C.

    Activity Assay:

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: We have previously shown that these two katanin constructs have similar high severing activity (Bailey et al., ). .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon.

    Expressing:

    Article Title: Multivalent Antiviral XTEN–Peptide Conjugates with Long in Vivo Half-Life and Enhanced Solubility
    Article Snippet: .. XTEN Expression All of the thiol-containing XTEN precursors (XTEN-1, 2, 3, 4) were expressed in the BL21 E. coli strain (New England Biolabs, #C2530H) using 5 L B. Braun Biostat B glass-jacketed fermentation vessels. .. 125 mL starter cultures were used to inoculate 1.7 L batches of fermentation media containing 50 mM (NH4 )2 SO4 , 20 mM K2 HPO4 , 15 mM KH2 PO4 , 4.5 mM C6 H5 Na3 O7 ·2H2 O, 11 mM NaH2 PO4 , 10 mM MgSO4 , 30 g/L NZ BL4 soy peptone (Kerry Bioscience, #5X00043), 15 g/L yeast extract (Teknova, #Y9020), 0.25 mL/L polypropylene glycol 2000, trace elements, and 10 mg/mL tetracycline.

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: .. The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. Escherichia coli BL21 was cultured in 10 mL of Luria–Bertani (LB) medium at 37 °C overnight; 0.1 mL of the overnight culture was transferred into 50 mL of LB medium containing 50 µ m kanamycin and incubated at 37 °C until A 600 nm reached OD = 0.6.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Paragraph title: Expression and Purification of Wild-type and C8A Mutant RNF114 Protein ... Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions. .. The purified protein was used for rabbit immunizations (Eurogentech, Liège, Belgium).

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: Paragraph title: Generation and expression of GST fusion proteins ... BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: An isopropyl β -D-1 thiogalactopyranoside-inducible expression system was used for expression and purification. .. The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA).

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: The control was made by sham-purification from the conditioned media from HEK cells that had not been transfected with a cDNA expressing the Myc-tagged netrin-1. .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA).

    Modification:

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: .. Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Proteins were purified by immobilized metal ion affinity chromatography with a 1 ml His-Trap column (Äkta pure system, GE healthcare, UK) with buffer A: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 30 mM imidazole and a gradient of 0–100 % buffer B: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 500 mM imidazole.

    Transformation Assay:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice. ..

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: .. The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. Escherichia coli BL21 was cultured in 10 mL of Luria–Bertani (LB) medium at 37 °C overnight; 0.1 mL of the overnight culture was transferred into 50 mL of LB medium containing 50 µ m kanamycin and incubated at 37 °C until A 600 nm reached OD = 0.6.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H). .. The following day, a single transformed colony was used to inoculate 50 mL of nutrient rich LB medium containing kanamycin (50 μg/mL) and was incubated at 37 °C overnight, with agitation (250 rpm).

    Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin
    Article Snippet: .. GST Pull-Down Assays The GST-SMAD4 fusion protein was induced with 0.1 mM IPTG in BL21 (C2530H) Escherichia coli competent cells (New England BioLabs, NEB, MA, United States) ) transformed with pGEX-2TK-SMAD4 and after 4 h, the bacteria were lysed in 2% sarkosyl-STE buffer (10 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) and sonicated. .. HEK293-I3A cells were transfected with pCMV-HA-MyD88 or one of the three pCMV-HA-MyD88 deletion constructs.

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon. ..

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: .. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced. .. Glutathione S-transferase pull-down Glutathione-Sepharose-4B beads (30 μl) (GE Healthcare Life Science, Pittsburgh, PA) were conjugated with GST (expressed protein from a 250 μl bacterial pellet per 30 μl of beads) or recombinant GST-tagged (expressed protein from a 3 ml bacterial pellet per 30 μl of beads) [ ] and incubated with lysate from 2×107 Caco-2 or SW620 cells (1500 μg protein) or purified Akt1 (0.35 μg) (Origene, Rockville, MD) overnight at 4°C.

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: .. The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA). ..

    Article Title: Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria
    Article Snippet: .. Protein purification For purification of IolG and IdhA, pOPS0141 and pOPS0142 were transformed into BL21-competent E. coli (New England Biolabs) and grown overnight in 5 ml LBAmp . ..

    Transfection:

    Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin
    Article Snippet: GST Pull-Down Assays The GST-SMAD4 fusion protein was induced with 0.1 mM IPTG in BL21 (C2530H) Escherichia coli competent cells (New England BioLabs, NEB, MA, United States) ) transformed with pGEX-2TK-SMAD4 and after 4 h, the bacteria were lysed in 2% sarkosyl-STE buffer (10 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) and sonicated. .. HEK293-I3A cells were transfected with pCMV-HA-MyD88 or one of the three pCMV-HA-MyD88 deletion constructs.

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: The control was made by sham-purification from the conditioned media from HEK cells that had not been transfected with a cDNA expressing the Myc-tagged netrin-1. .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA).

    Ligation:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice. ..

    Protease Inhibitor:

    Article Title: RAB21 Activity Assay Using GST-fused APPL1
    Article Snippet: .. 50 ml conical tube (Sarstedt AG & CO, catalog number: 62.547.004) 100 mm Tissue Culture Dish (Corning, catalog number: 430167) 0.22 μm filtering unit (Genesee Scientific Corporation, catalog number: 25-227) Cell lifter (Corning, catalog number: 3008) Escherichia coli BL21 (New England Biolabs, catalog number: C2530H) pGEX-5X-3 (GE Healthcare, catalog number: 27-4586-01) pGEX5X3-Happl1 (aa 5-419) (self made) ( ) pAcEGFP-C1 vector (Clontech, catalog number: 632470) pEGFP-C1:RAB21 wild type [human RAB21 cloned in pAcEGFP-C1 (this construct was used to generate the stable HeLa M cell line)] (self made) ( ) EGFP:RAB21wt (wildtype) stably-transfected HeLa M cells (self made) ( ) Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Biopioneer, catalog number: c0012) Ampicillin Sodium Salt (Crystalline Powder) (Thermo Fisher Scientific, catalog number: ) BD Bacto™ Tryptone (Thermo Fisher Scientific, catalog number: DF0123173) Yeast Extract (Thermo Fisher Scientific, catalog number: 212750) Glutathione Sepharose 4B beads (GE Healthcare, catalog number: 17-0756-01) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-5) Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml) Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15140-122”. .. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200-056) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25200-056”.

    Cell Culture:

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. Escherichia coli BL21 was cultured in 10 mL of Luria–Bertani (LB) medium at 37 °C overnight; 0.1 mL of the overnight culture was transferred into 50 mL of LB medium containing 50 µ m kanamycin and incubated at 37 °C until A 600 nm reached OD = 0.6.

    Generated:

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: Double-stranded (ds) RNAs for intererence in S2 cells have been generated using the following primers: T3-Snap29 5′-TAATACGACTCACTATAGGGAGA AACCCAGGAGGTGGGTAAG-3′ T7- Snap29 5′-AATTAACCCTCACTAAAGGGAGA ATGTTATCCAGCAATTCATTTTG-3′ DsRNA were in vitro transcribed with the T3 (Promega, P208C) and T7 (Promega, P207B) polymerase according to manufacturer's instructions, annealed, and incubated with the cells at a final concentration of 15ug/106 cells for 72 h. Multiple sequence analysis was performed using ClustalX using standard parameters. .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Droplet Countercurrent Chromatography:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA). .. The fusion protein was eluted with maltose and analyzed by SDS-PAGE, followed by Coomassie Blue staining.

    Sequencing:

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: .. The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. Escherichia coli BL21 was cultured in 10 mL of Luria–Bertani (LB) medium at 37 °C overnight; 0.1 mL of the overnight culture was transferred into 50 mL of LB medium containing 50 µ m kanamycin and incubated at 37 °C until A 600 nm reached OD = 0.6.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H). .. The following day, a single transformed colony was used to inoculate 50 mL of nutrient rich LB medium containing kanamycin (50 μg/mL) and was incubated at 37 °C overnight, with agitation (250 rpm).

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: Double-stranded (ds) RNAs for intererence in S2 cells have been generated using the following primers: T3-Snap29 5′-TAATACGACTCACTATAGGGAGA AACCCAGGAGGTGGGTAAG-3′ T7- Snap29 5′-AATTAACCCTCACTAAAGGGAGA ATGTTATCCAGCAATTCATTTTG-3′ DsRNA were in vitro transcribed with the T3 (Promega, P208C) and T7 (Promega, P207B) polymerase according to manufacturer's instructions, annealed, and incubated with the cells at a final concentration of 15ug/106 cells for 72 h. Multiple sequence analysis was performed using ClustalX using standard parameters. .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: Plasmids were purified via MiniPrep (QIAGEN, Valencia, CA) before sequencing. .. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: The target sequence was inserted into pAVU6+27 between Sal I site and Xba I site. .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA).

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: .. Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Proteins were purified by immobilized metal ion affinity chromatography with a 1 ml His-Trap column (Äkta pure system, GE healthcare, UK) with buffer A: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 30 mM imidazole and a gradient of 0–100 % buffer B: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 500 mM imidazole.

    Sonication:

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. The pellet was resuspended in phosphate‐buffered saline (PBS) and sonicated on ice.

    Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin
    Article Snippet: .. GST Pull-Down Assays The GST-SMAD4 fusion protein was induced with 0.1 mM IPTG in BL21 (C2530H) Escherichia coli competent cells (New England BioLabs, NEB, MA, United States) ) transformed with pGEX-2TK-SMAD4 and after 4 h, the bacteria were lysed in 2% sarkosyl-STE buffer (10 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) and sonicated. .. HEK293-I3A cells were transfected with pCMV-HA-MyD88 or one of the three pCMV-HA-MyD88 deletion constructs.

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon. .. Cells were pelleted and lysed in resuspension buffer (20 mM Hepes pH 7.7, 250 mM NaCl, 0.5 mM BME, 10% glycerol, 0.25 mM ATP) via sonication.

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA). .. The culture was allowed to continue to grow at 16°C for 16 h. The cells were lysed in resuspension buffer (20 mM HEPES-HCl, pH 7.7, 250 mM NaCl, 0.5 mM β -mercaptoethanol, 10% glycerol, and 0.25 mM ATP) via sonication.

    Affinity Purification:

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions. .. Sera were affinity purified using AminoLink® Kit (Pierce Biotechnology, 44890).

    Binding Assay:

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: For data comparing depolymerization rates as a function of the concentration of katanin and quantifying katanin binding, we used a human p60 isoform with a green fluorescent protein (GFP) tag and maltose binding protein (MBP) tag for purification. .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon.

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: We also made an optimized human p60 construct with a maltose binding protein and GFP (GeneWiz, Cambridge, MA). .. The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA).

    Mutagenesis:

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Paragraph title: Expression and Purification of Wild-type and C8A Mutant RNF114 Protein ... Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: Point mutations (L113A, P116C, P116G, P116N, P117K, and P116S) and triple mutants (L113A/P116N/P117K, L113A/P116C/P117G, L113A/P116A/P117A) were generated using the Quick Change II XL Site-Directed Mutagenesis kit (Agilent Technologies, (Santa Clara, CA). .. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.

    Labeling:

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Purified proteins were desalted by gel filtration with Zeba spin columns (7000 MWCO, Thermo Fischer Scientific, USA) and subsequently labeled with ten-fold molar excess of Alexa647-NHS (Thermo Fischer Scientific, USA) in 0.1 mM KH2 PO4 buffer at RT overnight.

    Purification:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: Purified, digested insert and vector were ligated with the following conditions 1 μL 10X T4 DNA ligase buffer (NEB), 50 ng vector, 150 ng insert, 0.5 μL T4 DNA ligase (NEB) in 10 μL total for 10 min at room temperature. .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Paragraph title: Expression and Purification of Wild-type and C8A Mutant RNF114 Protein ... Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions. .. The purified protein was used for rabbit immunizations (Eurogentech, Liège, Belgium).

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: For data comparing depolymerization rates as a function of the concentration of katanin and quantifying katanin binding, we used a human p60 isoform with a green fluorescent protein (GFP) tag and maltose binding protein (MBP) tag for purification. .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon.

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: Plasmids were purified via MiniPrep (QIAGEN, Valencia, CA) before sequencing. .. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: An isopropyl β -D-1 thiogalactopyranoside-inducible expression system was used for expression and purification. .. The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA).

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA). .. The fusion protein was eluted with maltose and analyzed by SDS-PAGE, followed by Coomassie Blue staining.

    Article Title: Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria
    Article Snippet: .. Protein purification For purification of IolG and IdhA, pOPS0141 and pOPS0142 were transformed into BL21-competent E. coli (New England Biolabs) and grown overnight in 5 ml LBAmp . ..

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Proteins were purified by immobilized metal ion affinity chromatography with a 1 ml His-Trap column (Äkta pure system, GE healthcare, UK) with buffer A: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 30 mM imidazole and a gradient of 0–100 % buffer B: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 500 mM imidazole.

    Protein Purification:

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: Paragraph title: Protein purification ... The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA).

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: Paragraph title: Protein purification ... We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon.

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: Paragraph title: Protein purification ... The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA).

    Article Title: Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria
    Article Snippet: .. Protein purification For purification of IolG and IdhA, pOPS0141 and pOPS0142 were transformed into BL21-competent E. coli (New England Biolabs) and grown overnight in 5 ml LBAmp . ..

    Polymerase Chain Reaction:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: PCR products were column purified (Qiagen PCR cleanup kit #28104), and 2 μg of DNA was digested with EcoRI-HF and PstI-HF (NEB) with the manufacturer's recommended conditions, followed by another PCR cleanup (Qiagen #28104). .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Gibson Assembly (NEB Gibson Assembly 2× Master Mix) was used to assemble the purified PCR product into the linearized vector. .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: The PCR product was inserted using BamHI and XhoI into pGEX -GST (Addgene Vector Database, 27- 4597- 01). .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: PCR products were introduced into the pGEX-4T1 template between 5′ EcoRI and 3′XhoI sites. .. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.

    Affinity Chromatography:

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Proteins were purified by immobilized metal ion affinity chromatography with a 1 ml His-Trap column (Äkta pure system, GE healthcare, UK) with buffer A: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 30 mM imidazole and a gradient of 0–100 % buffer B: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 500 mM imidazole.

    Blocking Assay:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: G-blocks were PCR amplified using Forward primer 5′-TTATTCGAATTCGCGGCCGCTTCTAGAG and Reverse primer 5′-GGATTTCTGCAGCGGCCGCTACTAGTA with the following conditions: in a 50 μL reaction: 10 μL 5x HF buffer (New England Biolabs, NEB #E0553L), 2 μL 10 mM dNTP mix (NEB #N0447L), 1 μL 100 μM forward and reverse primer, 25 ng g block template, and 0.5 μL phusion polymerase. .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    Recombinant:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA). .. The fusion protein was eluted with maltose and analyzed by SDS-PAGE, followed by Coomassie Blue staining.

    Gel Extraction:

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: Products for PCR were assessed using 1% agarose gels (Invitrogen), and a QIAquick Gel Extraction kit (Qiagen) was used to purify PCR products of the correct length. .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H).

    SDS Page:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA). .. The fusion protein was eluted with maltose and analyzed by SDS-PAGE, followed by Coomassie Blue staining.

    Plasmid Preparation:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: Paragraph title: Construction of Plasmid DNA ... The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: .. The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. Escherichia coli BL21 was cultured in 10 mL of Luria–Bertani (LB) medium at 37 °C overnight; 0.1 mL of the overnight culture was transferred into 50 mL of LB medium containing 50 µ m kanamycin and incubated at 37 °C until A 600 nm reached OD = 0.6.

    Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation
    Article Snippet: .. Kanamycin (Kan)-resistant colonies were grown in LB media, and a Miniprep (Qiagen) kit was used to isolate the plasmid before sequence verification with appropriate primers. pET24a His8 –MBP plasmid (100 ng) containing the desired RNF114 construct was transformed into chemically competent E. coli BL21(DE3) cells (NEB product # C2530H). .. The following day, a single transformed colony was used to inoculate 50 mL of nutrient rich LB medium containing kanamycin (50 μg/mL) and was incubated at 37 °C overnight, with agitation (250 rpm).

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: The PCR product was inserted using BamHI and XhoI into pGEX -GST (Addgene Vector Database, 27- 4597- 01). .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Article Title: Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor
    Article Snippet: Strains, plasmids and media Escherichia coli (E. coli ) DH5α was used for routine cloning and plasmid propagation (NEB, #C2987I). .. E. coli BL21 strain (NEB, #C2530H) was used as production strain harboring the original pathway eAKP672.

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon. ..

    Article Title: Inhibition of pressure-activated cancer cell adhesion by FAK-derived peptides
    Article Snippet: Generation and expression of GST fusion proteins Bacterial expression vector pGEX-4T1 (GE Healthcare, Munich, Germany) was used as a template to generate mutated and truncated human FAK as GST (Glutathione S-transferase) fusion proteins. .. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.

    Article Title: Proteome-wide enrichment of proteins modified by lysine methylation
    Article Snippet: Plasmids: 3×MBT-pGEX6P-1 and 3×MBTD355N -pGEX6P-1 constructs (AddGene plasmid nos. .. 46987 and 46988, respectively) Mammalian SILAC medium: DMEM (Thermo Scientific/Pierce, cat. no. 88420) and RPMI (Thermo Scientific/Pierce, cat. no. 88421) Dialyzed FBS for SILAC (Thermo Scientific/Pierce, cat. no. 88440) Yeast SILAC medium (based on Kubota et al. ): Dropout mix minus leucine, lysine, arginine and adenine (US Biological, cat. no. D9515F) Yeast nitrogen base without amino acids (Sigma, cat. no. Y0626) Leucine (Sigma, cat. no. L8912) Adenine (Sigma, cat. no. A8626) Glucose (Invitrogen-Gibco, cat. no. 15023021) SILAC amino acids: L-lysine-2HCl (Thermo Scientific/Pierce, cat. no. 88429), L-arginine-HCl (Thermo Scientific/Pierce, cat. no. 88427), 2 H4 -L-lysine-2HCl (Thermo Scientific/Pierce, cat. no. 88438), 13 C6 -L-arginine-HCl (Thermo Scientific/Pierce, cat. no. 88433), 15 N2 13 C6 -L-lysine-2HCl (Thermo Scientific/Pierce, cat. no. 88209), 15 N4 13 C6 -L-arginine-HCl (Thermo Scientific/Pierce, cat. no. 89990), L-proline (Thermo Scientific/Pierce, cat. no. 88430) Yeast strain from any background (for example, Fisher cat. no. NC0537738), but they must be auxotrophic for arginine and lysine ( arg4 lys2 ) as described in ref. LB medium (Sigma, cat. no. L3522) BL21-competent Escherichia coli (New England BioLabs, cat. no. C2530 H) Ampicillin sodium salt (Sigma, cat. no. A0166) IPTG (Sigma, cat. no. I5502) Tris base (Fisher, cat. no. BP152) Hydrochloric acid (HCl; Fisher, cat. no. A144) !

    Article Title: Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
    Article Snippet: .. The plasmid was transformed into BL21 competent Escherichia coli (New England BioLabs, Ipswich, MA). ..

    Article Title: RAB21 Activity Assay Using GST-fused APPL1
    Article Snippet: .. 50 ml conical tube (Sarstedt AG & CO, catalog number: 62.547.004) 100 mm Tissue Culture Dish (Corning, catalog number: 430167) 0.22 μm filtering unit (Genesee Scientific Corporation, catalog number: 25-227) Cell lifter (Corning, catalog number: 3008) Escherichia coli BL21 (New England Biolabs, catalog number: C2530H) pGEX-5X-3 (GE Healthcare, catalog number: 27-4586-01) pGEX5X3-Happl1 (aa 5-419) (self made) ( ) pAcEGFP-C1 vector (Clontech, catalog number: 632470) pEGFP-C1:RAB21 wild type [human RAB21 cloned in pAcEGFP-C1 (this construct was used to generate the stable HeLa M cell line)] (self made) ( ) EGFP:RAB21wt (wildtype) stably-transfected HeLa M cells (self made) ( ) Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Biopioneer, catalog number: c0012) Ampicillin Sodium Salt (Crystalline Powder) (Thermo Fisher Scientific, catalog number: ) BD Bacto™ Tryptone (Thermo Fisher Scientific, catalog number: DF0123173) Yeast Extract (Thermo Fisher Scientific, catalog number: 212750) Glutathione Sepharose 4B beads (GE Healthcare, catalog number: 17-0756-01) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-5) Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml) Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15140-122”. .. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200-056) Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25200-056”.

    Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side
    Article Snippet: .. Production of titin-based tension probes Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. .. Proteins were purified by immobilized metal ion affinity chromatography with a 1 ml His-Trap column (Äkta pure system, GE healthcare, UK) with buffer A: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 30 mM imidazole and a gradient of 0–100 % buffer B: KH2 PO4 buffer (pH 7.4) + 1 mM DTT + 500 mM imidazole.

    shRNA:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: TUBB3 shRNA and control shRNA were gifts from David L. Turner , EB3-GFP constructs were from Niels Galjart and TUBB3-V5 were from Elizabeth C Engle. .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli
    Article Snippet: Six micrograms of backbone vector pSB1A3 was digested with EcoRI-HF and PstI-HF (NEB), electrophoresed on a 1% agarose gel for 45 min, and gel extracted (Sigma #NA1111-1KT). .. The ligation was transformed into chemically competent BL21 E. coli (NEB #C2530H) with the following conditions: 5 μL of ligation into 50 μL cells on ice for 5 min, followed by 45 s at 42°C, followed by 2 min on ice.

    In Vitro:

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: Double-stranded (ds) RNAs for intererence in S2 cells have been generated using the following primers: T3-Snap29 5′-TAATACGACTCACTATAGGGAGA AACCCAGGAGGTGGGTAAG-3′ T7- Snap29 5′-AATTAACCCTCACTAAAGGGAGA ATGTTATCCAGCAATTCATTTTG-3′ DsRNA were in vitro transcribed with the T3 (Promega, P208C) and T7 (Promega, P207B) polymerase according to manufacturer's instructions, annealed, and incubated with the cells at a final concentration of 15ug/106 cells for 72 h. Multiple sequence analysis was performed using ClustalX using standard parameters. .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Produced:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: .. Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA). .. The fusion protein was eluted with maltose and analyzed by SDS-PAGE, followed by Coomassie Blue staining.

    Concentration Assay:

    Article Title: Functional characterization of the citrus canker susceptibility gene CsLOB1
    Article Snippet: The expression vector pGEX‐4T‐1‐CsLOB1 was confirmed by sequencing, and transformed into BL21 competent Escherichia coli (New England Biolabs, Ipswich, MA, USA). .. A final concentration of 0.25 m m of isopropylthiogalactopyranoside (IPTG) was added to the LB medium.

    Article Title: Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
    Article Snippet: Double-stranded (ds) RNAs for intererence in S2 cells have been generated using the following primers: T3-Snap29 5′-TAATACGACTCACTATAGGGAGA AACCCAGGAGGTGGGTAAG-3′ T7- Snap29 5′-AATTAACCCTCACTAAAGGGAGA ATGTTATCCAGCAATTCATTTTG-3′ DsRNA were in vitro transcribed with the T3 (Promega, P208C) and T7 (Promega, P207B) polymerase according to manufacturer's instructions, annealed, and incubated with the cells at a final concentration of 15ug/106 cells for 72 h. Multiple sequence analysis was performed using ClustalX using standard parameters. .. Antibody production GST-Snap29 expression was carried out in the E. coli BL21 strain (NEB, C2530H) upon IPTG induction and gluthatione-Sepharose beads (Invitrogen, 10- 1243) purification was performed under standard conditions.

    Article Title: Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails. Katanin catalyzes microtubule depolymerization independently of tubulin C‐terminal tails
    Article Snippet: For data comparing depolymerization rates as a function of the concentration of katanin and quantifying katanin binding, we used a human p60 isoform with a green fluorescent protein (GFP) tag and maltose binding protein (MBP) tag for purification. .. We transformed the plasmid into BL21 competent Escherichia coli (New England BioLabs, Waltham, MA), grew a 5 mL liquid culture for 3–4 hr, and added it to a 400 mL culture in the afternoon.

    Staining:

    Article Title: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance
    Article Snippet: Recombinant DCC intracellular domain tagged with maltose-binding protein (DCC-ICD-MBP) was produced from BL21 competent E. coli and purified using amylose resin (New England Biolabs, Ipswich, MA, USA). .. The fusion protein was eluted with maltose and analyzed by SDS-PAGE, followed by Coomassie Blue staining.

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    New England Biolabs competent bl21 e coli
    Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21  E. coli  and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;
    Competent Bl21 E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/competent bl21 e coli/product/New England Biolabs
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    Price from $9.99 to $1999.99
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    Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21  E. coli  and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;

    Journal: Journal of Clinical Microbiology

    Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

    doi: 10.1128/JCM.03491-14

    Figure Lengend Snippet: Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21 E. coli and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;

    Article Snippet: Chemically competent BL21 E. coli (NEB) was transformed with 10 ng pMAL-c5e or pMAL-c3B for overexpression of recombinant MBP or MBP-c3B fusion protein, according to the manufacturer's instructions.

    Techniques: Recombinant, Expressing, Purification, Staining, Affinity Purification