10 beta electrocompetent cells  (New England Biolabs)


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    Name:
    NEB 10 beta Electrocompetent E coli
    Description:
    NEB 10 beta Electrocompetent E coli 6x0 1 ml
    Catalog Number:
    C3020K
    Price:
    214
    Size:
    0 6 ml
    Category:
    Competent Bacteria
    Score:
    85
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    New England Biolabs 10 beta electrocompetent cells
    NEB 10 beta Electrocompetent E coli
    NEB 10 beta Electrocompetent E coli 6x0 1 ml
    https://www.bioz.com/result/10 beta electrocompetent cells/product/New England Biolabs
    Average 96 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    10 beta electrocompetent cells - by Bioz Stars, 2020-01
    96/100 stars

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    Clone Assay:

    Article Title: Antisense transcription as a tool to tune gene expression
    Article Snippet: Plasmid backbones were then digested with the same restriction enzymes and cleaned using DNA Clean & Concentrator columns. .. After digestion, the library inserts and plasmid backbones were ligated using T4 DNA ligase (New England Biolabs M0208) and cloned into E. coli NEB10β‐electrocompetent cells (New England Biolabs C3020K), resulting in three libraries (PhlF, SrpR, and TarA) of ~160,000 clones each and > 20‐fold coverage of the designed sequence space. .. Each library was scraped from solid media plates and frozen at −80°C in 200 μl aliquots with 15% glycerol for subsequent analysis.

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: After a 10-day incubation, HeLa-RC cell clones derived from each G418-resistant colony produced by pLEmH, TK109-17 or Nb2A3-2 were cultured separately until the total number of cells reached ∼1 × 106 cells per clone. .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: In this study, the localization, biochemical characteristics, and physiological role of two SLH domain enzymes from C.kronotskyensis , xylanase Calkro_0402 and laminarinase Calkro_0111, were examined from this perspective. .. Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. Axenic strains of C. bescii and C. kronotskyensis were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures.

    Article Title: New CRISPR-Cas systems from uncultivated microbes
    Article Snippet: Putative targets identified from metagenomic sequence analysis or PAM depletion assays were cloned into a pUC19 plasmid. .. 10 ng of target plasmid were transformed into electrocompetent E. coli (NEB Stable) containing the CRISPR loci plasmid.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Plasmid 44251 digested with SpeI and ApaI (New England Biolabs) was used as the cloning backbone. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The library oligonucleotides were then cloned downstream of the human U6 promoter in a lentiviral vector containing EGFP downstream of the human PGK promoter (pLKO.1-EGFP). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: This double-stranded DNA was ligated into the kanamycin-resistant plasmid attB_GPiM upstream of the IRES element using directional cloning with the EcoRI-HF and SacII restriction enzymes (New England Biolabs). .. The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count.

    Centrifugation:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: After addition of Pico-Break 1, the samples were vortexed, followed by centrifugation (15 s, 2,000 r.c.f.) to attain phase separation. .. Electrocompetent E. coli cells (50 μl, β-10 E. coli cells, NEB) were transformed with 5 μl of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser).

    Amplification:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: A genome-wide lentiviral CRISPR gRNA library was utilised that contains 87,897 gRNAs targeting 19,150 mouse protein-coding genes , with up to five gRNAs per gene (Addgene: #50947). .. The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol. .. Briefly, E. coli were transformed via high-efficiency electroporation, to ensure faithful library replication, and incubated at 37 °C for 1 h with SOC recovery medium (ThermoFisher) before growing in 500 ml 2xTY (16 g/l Tryptone, 10 g/l Yeast Extract, 5.0 g/l NaCl) + ampicillin (50 µg/ml) medium at 37 °C overnight with 230 rpm shaking.

    Article Title: Antisense transcription as a tool to tune gene expression
    Article Snippet: Plasmid backbones encoding repressor protein‐based NOT gates (PhlF, SrpR, TarA; maps in ) were amplified by PCR with primers to add NotI and SbfI restriction sites to the 3′‐end of the repressor gene. .. After digestion, the library inserts and plasmid backbones were ligated using T4 DNA ligase (New England Biolabs M0208) and cloned into E. coli NEB10β‐electrocompetent cells (New England Biolabs C3020K), resulting in three libraries (PhlF, SrpR, and TarA) of ~160,000 clones each and > 20‐fold coverage of the designed sequence space.

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: Following amplification, the tile PCR products were incorporated into the appropriate linear pYES2-PTEN by SLiCE-mediated recombination. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. C. bescii strain JWCB018 ( ) and non-replicating vector pDCW121 ( ) were obtained from J. Westpheling (University of Georgia, Athens, Georgia).

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: The phylogenetic group was determined by PCR [ ], the class I integron was amplified and the purified amplification products were sequenced as described previously [ ]. .. Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany).

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: Paragraph title: Barcode library generation, two-step amplification, sequencing and data analysis ... The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count.

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Each fragment was individually amplified using three sets of unique primers (P1.For, P1.Rev, P2.For, P2.Rev, P3.For, P3.Rev) with an optimized number of PCR cycles determined by quantitative PCR analysis to prevent over-amplification. .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA).

    Article Title: Automated multiplex genome-scale engineering in yeast
    Article Snippet: The CAD strain was constructed previously via integration of an RNAi pathway into the CEN.PK2-1c genome. .. Zymo 5α Z-competent E. coli (Zymo Research, Irvine, CA) and NEB 10β Electrocompetent E. coli (New England Biolabs, Ipswich, MA) were used for plasmid amplification and library construction, respectively. .. S. cerevisiae strains were cultivated in either synthetic complete (SC) dropout medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate and 0.083% amino-acid dropout mix, 0.01% adenine hemisulfate and 2% glucose) or YPAD medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate and 2% glucose).

    Depletion Assay:

    Article Title: Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
    Article Snippet: Paragraph title: PAM depletion assay ... Electrocompetent NEB 10-beta cells (New England Biolabs) were electroporated with two plasmids.

    Synthesized:

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: These DNA tiles were synthesized as 130-mers (prefix: PTENTile) as part of a 12,000-feature oligo pool by CustomArray. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Molecular biology reagents: DNA for sc-(V1V2)3 and gp70 V1V2 constructs were purchased as gBlock gene fragments from Integrated DNA Technologies (IDT), or synthesized by Genewiz, Inc. All oligonucleotides were purchased from IDT. .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning.

    Construct:

    Article Title: Antisense transcription as a tool to tune gene expression
    Article Snippet: The oligonucleotide library was constructed by CustomArray, Inc., using their CMOS semiconductor technology. .. After digestion, the library inserts and plasmid backbones were ligated using T4 DNA ligase (New England Biolabs M0208) and cloned into E. coli NEB10β‐electrocompetent cells (New England Biolabs C3020K), resulting in three libraries (PhlF, SrpR, and TarA) of ~160,000 clones each and > 20‐fold coverage of the designed sequence space.

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. Genes of interest were amplified by polymerase chain reaction (PCR) and cloned using the pET46 Ek/LIC vector kit (EMD Millipore) for protein expression.

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Molecular biology reagents: DNA for sc-(V1V2)3 and gp70 V1V2 constructs were purchased as gBlock gene fragments from Integrated DNA Technologies (IDT), or synthesized by Genewiz, Inc. All oligonucleotides were purchased from IDT. .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Unique sgRNA targets were constructed by PCR amplifying cloning inserts (primers obtained from Integrated DNA Technologies) replacing the RFP target sequence with the new target sequence for each gene. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Article Title: Automated multiplex genome-scale engineering in yeast
    Article Snippet: The CAD strain was constructed previously via integration of an RNAi pathway into the CEN.PK2-1c genome. .. Zymo 5α Z-competent E. coli (Zymo Research, Irvine, CA) and NEB 10β Electrocompetent E. coli (New England Biolabs, Ipswich, MA) were used for plasmid amplification and library construction, respectively.

    Real-time Polymerase Chain Reaction:

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs). .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Each fragment was individually amplified using three sets of unique primers (P1.For, P1.Rev, P2.For, P2.Rev, P3.For, P3.Rev) with an optimized number of PCR cycles determined by quantitative PCR analysis to prevent over-amplification. .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA).

    Incubation:

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: SLiCE reactions were 10 μL and consisted of 100 ng of linearized vector with 15 ng of tile DNA, along with 1× SLiCE buffer and 1× SLiCE extract. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli . .. Transformation reactions were plated on LB agar plates with 100 mg/mL carbenecillin (GoldBio) and grown overnight at 37°C.

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: After a 10-day incubation, HeLa-RC cell clones derived from each G418-resistant colony produced by pLEmH, TK109-17 or Nb2A3-2 were cultured separately until the total number of cells reached ∼1 × 106 cells per clone. .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Electrocompetent E. coli cells (50 μl, β-10 E. coli cells, NEB) were transformed with 5 μl of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser). .. Electrocompetent E. coli cells (50 μl, β-10 E. coli cells, NEB) were transformed with 5 μl of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser).

    Article Title: Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
    Article Snippet: Electrocompetent NEB 10-beta cells (New England Biolabs) were electroporated with two plasmids. .. The second plasmid contains the target protospacer and a kanamycin resistance gene.

    Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
    Article Snippet: A total of ten electroporations were performed according to the manufacturer’s protocol using 1 ng of ligated vector and 25 ul of DH10beta E. coli Electrocompetent Cells (NEB). .. Each electroporation reaction was then plated onto two 15-cm diameter agar plates containing Luria broth medium (Life Technologies) and 100 μg/ml carbenicillin.

    Expressing:

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: In this study, the localization, biochemical characteristics, and physiological role of two SLH domain enzymes from C.kronotskyensis , xylanase Calkro_0402 and laminarinase Calkro_0111, were examined from this perspective. .. Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. Axenic strains of C. bescii and C. kronotskyensis were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Plasmid pPO-dCas9ω was constructed in a previous study (Otoupal et al., ) and used for expression of dCas9-ω alongside 44251. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The full-length cassette was then assembled by combining 15 ng of each fragment and DNA primers P1.For and P3.Rev into a single PCR reaction. .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA). .. Polymerase variants were grown as a population of E. coli carrying the pGDR11 plasmid encoding the polymerase of interest in Luria–Bertani (LB) broth supplemented with ampicillin (100 μg ml−1 ).

    Article Title: Automated multiplex genome-scale engineering in yeast
    Article Snippet: Zymo 5α Z-competent E. coli (Zymo Research, Irvine, CA) and NEB 10β Electrocompetent E. coli (New England Biolabs, Ipswich, MA) were used for plasmid amplification and library construction, respectively. .. S. cerevisiae strains were cultivated in either synthetic complete (SC) dropout medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate and 0.083% amino-acid dropout mix, 0.01% adenine hemisulfate and 2% glucose) or YPAD medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate and 2% glucose).

    Genome Wide:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: A genome-wide lentiviral CRISPR gRNA library was utilised that contains 87,897 gRNAs targeting 19,150 mouse protein-coding genes , with up to five gRNAs per gene (Addgene: #50947). .. The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol.

    Transformation Assay:

    Article Title: New CRISPR-Cas systems from uncultivated microbes
    Article Snippet: Putative targets identified from metagenomic sequence analysis or PAM depletion assays were cloned into a pUC19 plasmid. .. 10 ng of target plasmid were transformed into electrocompetent E. coli (NEB Stable) containing the CRISPR loci plasmid. .. CasX.1 was used for the plasmid interference assays under control of native promoters or using a strong heterologous promoter (J23119) for sgRNA and crRNA expression.

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The DNA Clean & Concentrator-5 also facilitates removal of protein from the sample. .. Electrocompetent E. coli cells (50 μl, β-10 E. coli cells, NEB) were transformed with 5 μl of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser). .. Sterile S.O.C.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Plasmid 44251 digested with SpeI and ApaI (New England Biolabs) was used as the cloning backbone. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs). .. Final constructs were recovered using Zyppy Plasmid Miniprep Kit (Zymo Research Corporation) and confirmed by sequencing (via GENEWIZ) before transformation into chemically competent E. coli MG1655 (ATCC 700926) harboring either dCas9 or dCas9-ω plasmids for gene repression or activation respectively.

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: Ligation was performed using Quick Ligase kit (New England Biolabs). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs). .. The pool of plasmids was prepared for infection using an endotoxin-free Maxi prep kit (Qiagen).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The full-length cassette was then assembled by combining 15 ng of each fragment and DNA primers P1.For and P3.Rev into a single PCR reaction. .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA). .. Polymerase variants were grown as a population of E. coli carrying the pGDR11 plasmid encoding the polymerase of interest in Luria–Bertani (LB) broth supplemented with ampicillin (100 μg ml−1 ).

    Derivative Assay:

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: After a 10-day incubation, HeLa-RC cell clones derived from each G418-resistant colony produced by pLEmH, TK109-17 or Nb2A3-2 were cultured separately until the total number of cells reached ∼1 × 106 cells per clone. .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Electroporation:

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: The digested DNA (∼20 μg) was then self-ligated overnight with T4 DNA ligase (350 U) in 500 μl volume at 16°C. .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml). .. Circular DNA containing a mneoI400 /ColE1 -marked L1.3, ZfL2-1 or Nimb2_DR insertion (with its flanking chicken or human genomic DNA) was isolated from the kanamycin-resistant cells.

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Transfection:

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count. .. The resulting plasmid was labeled attB-GPiM-N18-2M.

    Inverse PCR:

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: For each tile, we designed inverse PCR primers that linearized the pYES2-PTEN wild-type sequence, excluding the portion encoded by the corresponding tile. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Ligation:

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Phusion DNA Polymerase, Taq polymerase, DNA restriction and ligation enzymes were purchased from New England Biolabs (NEB), and DNA purification and gel-extraction were performed using Geneaid kits. .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning.

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: Ligation was performed using Quick Ligase kit (New England Biolabs). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Cell Culture:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol. .. Faithful replication/amplification of the library was confirmed by Illumina sequencing.

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: After a 10-day incubation, HeLa-RC cell clones derived from each G418-resistant colony produced by pLEmH, TK109-17 or Nb2A3-2 were cultured separately until the total number of cells reached ∼1 × 106 cells per clone. .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Generated:

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany). .. The size of the bla VIM-1 -containing plasmid was estimated by S1 nuclease pulsed-field gel electrophoresis (PFGE) [ ] using the following running conditions: 1–25 s, 17 h, 6 V/cm, 120 V. In addition, genomic DNA from the E. coli wild-type strain E-124–4 as well as the transformant TE-124–4 and the recipient strain NEB10-beta was isolated from overnight cultures of the selected isolates using the PureLink Genomic DNA Mini Kit (Invitrogen, Waltham, United States (US)).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The DNA cassette was generated from three gBlock fragments that were combined by overlapping PCR using AccuPrime DNA polymerase ( ). .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA).

    other:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The extracted aqueous phase was concentrated using a spin column (Zymo Research) and used to transform electrocompetent E. coli cells (β-10, NEB).

    Polymerase Chain Reaction:

    Article Title: Antisense transcription as a tool to tune gene expression
    Article Snippet: Plasmid backbones encoding repressor protein‐based NOT gates (PhlF, SrpR, TarA; maps in ) were amplified by PCR with primers to add NotI and SbfI restriction sites to the 3′‐end of the repressor gene. .. After digestion, the library inserts and plasmid backbones were ligated using T4 DNA ligase (New England Biolabs M0208) and cloned into E. coli NEB10β‐electrocompetent cells (New England Biolabs C3020K), resulting in three libraries (PhlF, SrpR, and TarA) of ~160,000 clones each and > 20‐fold coverage of the designed sequence space.

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: Following amplification, the tile PCR products were incorporated into the appropriate linear pYES2-PTEN by SLiCE-mediated recombination. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. C. bescii strain JWCB018 ( ) and non-replicating vector pDCW121 ( ) were obtained from J. Westpheling (University of Georgia, Athens, Georgia).

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: The phylogenetic group was determined by PCR [ ], the class I integron was amplified and the purified amplification products were sequenced as described previously [ ]. .. Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany).

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Unique sgRNA targets were constructed by PCR amplifying cloning inserts (primers obtained from Integrated DNA Technologies) replacing the RFP target sequence with the new target sequence for each gene. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of DpnI-digested and purified PCR product was ligated in 40 μl with 0.1 μl of concentrated T4 ligase (New England Biolabs) at 16°C overnight. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The PCR-amplified library was then purified (Qiagen), digested with BbsI (New England Biolabs) at 37 °C overnight, and purified by electrophoresis on a 2% agarose gel and recovered using gel extraction kit (Qiagen). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The full-length cassette was then assembled by combining 15 ng of each fragment and DNA primers P1.For and P3.Rev into a single PCR reaction. .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA). .. Polymerase variants were grown as a population of E. coli carrying the pGDR11 plasmid encoding the polymerase of interest in Luria–Bertani (LB) broth supplemented with ampicillin (100 μg ml−1 ).

    Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
    Article Snippet: Five reactions were combined and cleaned using a Qiaquick PCR purification Kit (Qiagen) and eluted into nuclease-free water. .. A total of ten electroporations were performed according to the manufacturer’s protocol using 1 ng of ligated vector and 25 ul of DH10beta E. coli Electrocompetent Cells (NEB).

    Recombinant:

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: In this study, the localization, biochemical characteristics, and physiological role of two SLH domain enzymes from C.kronotskyensis , xylanase Calkro_0402 and laminarinase Calkro_0111, were examined from this perspective. .. Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. Axenic strains of C. bescii and C. kronotskyensis were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures.

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count. .. HEK 293T TetBxb1BFP cells were transfected with pCAG-NLS-HA-Bxb1, followed the next day by attB-GPiM-N18-2M.

    Pulsed-Field Gel:

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany). .. The incompatibility (Inc-) group of the plasmid was determined by using the PCR-based replicon typing (PBRT) kit (Diatheva, Fano, Italy).

    Molecular Cloning:

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Site-directed mutagenesis was performed with Turbo pfu polymerase (Agilent). .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning. .. DNA plasmid purification from bacteria was performed using QIAPrep Spin Miniprep (Qiagen), Zyppy Plasmid Midiprep (Zymo Research) or E.Z.N.A.

    Mutagenesis:

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: Paragraph title: PTEN Saturation Mutagenesis ... Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Site-directed mutagenesis was performed with Turbo pfu polymerase (Agilent). .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning.

    Isolation:

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli . .. Transformation reactions were plated on LB agar plates with 100 mg/mL carbenecillin (GoldBio) and grown overnight at 37°C.

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: Genomic DNA was isolated from each clone using the GenElute mammalian genomic DNA miniprep kit (Sigma). .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: The phylogenetic group was determined by PCR [ ], the class I integron was amplified and the purified amplification products were sequenced as described previously [ ]. .. Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany). .. The incompatibility (Inc-) group of the plasmid was determined by using the PCR-based replicon typing (PBRT) kit (Diatheva, Fano, Italy).

    Subcloning:

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Flow Cytometry:

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count. .. HEK 293T TetBxb1BFP cells were transfected with pCAG-NLS-HA-Bxb1, followed the next day by attB-GPiM-N18-2M.

    Labeling:

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count. .. The library was grown in LB supplemented with 50 ng/μl kanamycin, and plasmid DNA was extracted with a Qiagen Midiprep Kit.

    Purification:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol. .. Briefly, E. coli were transformed via high-efficiency electroporation, to ensure faithful library replication, and incubated at 37 °C for 1 h with SOC recovery medium (ThermoFisher) before growing in 500 ml 2xTY (16 g/l Tryptone, 10 g/l Yeast Extract, 5.0 g/l NaCl) + ampicillin (50 µg/ml) medium at 37 °C overnight with 230 rpm shaking.

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: The phylogenetic group was determined by PCR [ ], the class I integron was amplified and the purified amplification products were sequenced as described previously [ ]. .. Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The DNA Clean & Concentrator-5 also facilitates removal of protein from the sample. .. Electrocompetent E. coli cells (50 μl, β-10 E. coli cells, NEB) were transformed with 5 μl of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser). .. Sterile S.O.C.

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of DpnI-digested and purified PCR product was ligated in 40 μl with 0.1 μl of concentrated T4 ligase (New England Biolabs) at 16°C overnight. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The vector backbone was digested with AgeI and EcoRI, treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), purified on a 1% agarose gel followed by gel extraction (Qiagen). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
    Article Snippet: Five reactions were combined and cleaned using a Qiaquick PCR purification Kit (Qiagen) and eluted into nuclease-free water. .. A total of ten electroporations were performed according to the manufacturer’s protocol using 1 ng of ligated vector and 25 ul of DH10beta E. coli Electrocompetent Cells (NEB).

    Sequencing:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol. .. Plasmid was purified from 500 ml bacterial cultures using an endotoxin-free plasmid maxi kit (Qiagen) as per manufacturer’s instructions.

    Article Title: Antisense transcription as a tool to tune gene expression
    Article Snippet: Plasmid backbones were then digested with the same restriction enzymes and cleaned using DNA Clean & Concentrator columns. .. After digestion, the library inserts and plasmid backbones were ligated using T4 DNA ligase (New England Biolabs M0208) and cloned into E. coli NEB10β‐electrocompetent cells (New England Biolabs C3020K), resulting in three libraries (PhlF, SrpR, and TarA) of ~160,000 clones each and > 20‐fold coverage of the designed sequence space. .. Each library was scraped from solid media plates and frozen at −80°C in 200 μl aliquots with 15% glycerol for subsequent analysis.

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: For each tile, we designed inverse PCR primers that linearized the pYES2-PTEN wild-type sequence, excluding the portion encoded by the corresponding tile. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: Paragraph title: Sequence analysis of LINE integrants ... Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany). .. Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany).

    Article Title: New CRISPR-Cas systems from uncultivated microbes
    Article Snippet: Putative targets identified from metagenomic sequence analysis or PAM depletion assays were cloned into a pUC19 plasmid. .. 10 ng of target plasmid were transformed into electrocompetent E. coli (NEB Stable) containing the CRISPR loci plasmid.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Unique sgRNA targets were constructed by PCR amplifying cloning inserts (primers obtained from Integrated DNA Technologies) replacing the RFP target sequence with the new target sequence for each gene. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: Paragraph title: Barcode library generation, two-step amplification, sequencing and data analysis ... The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count.

    CRISPR:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: Paragraph title: Lentiviral CRISPR screen ... The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol.

    Article Title: New CRISPR-Cas systems from uncultivated microbes
    Article Snippet: Putative targets identified from metagenomic sequence analysis or PAM depletion assays were cloned into a pUC19 plasmid. .. 10 ng of target plasmid were transformed into electrocompetent E. coli (NEB Stable) containing the CRISPR loci plasmid. .. CasX.1 was used for the plasmid interference assays under control of native promoters or using a strong heterologous promoter (J23119) for sgRNA and crRNA expression.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Paragraph title: CRISPR plasmid and strain construction ... Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Sample Prep:

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany). .. The size of the bla VIM-1 -containing plasmid was estimated by S1 nuclease pulsed-field gel electrophoresis (PFGE) [ ] using the following running conditions: 1–25 s, 17 h, 6 V/cm, 120 V. In addition, genomic DNA from the E. coli wild-type strain E-124–4 as well as the transformant TE-124–4 and the recipient strain NEB10-beta was isolated from overnight cultures of the selected isolates using the PureLink Genomic DNA Mini Kit (Invitrogen, Waltham, United States (US)).

    Plasmid Preparation:

    Article Title: Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
    Article Snippet: The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol. .. The gRNA library was amplified with NEB 10-beta electrocompetent Escherichia coli (NEB) as per the recommended protocol.

    Article Title: Antisense transcription as a tool to tune gene expression
    Article Snippet: Plasmid backbones were then digested with the same restriction enzymes and cleaned using DNA Clean & Concentrator columns. .. After digestion, the library inserts and plasmid backbones were ligated using T4 DNA ligase (New England Biolabs M0208) and cloned into E. coli NEB10β‐electrocompetent cells (New England Biolabs C3020K), resulting in three libraries (PhlF, SrpR, and TarA) of ~160,000 clones each and > 20‐fold coverage of the designed sequence space. .. Each library was scraped from solid media plates and frozen at −80°C in 200 μl aliquots with 15% glycerol for subsequent analysis.

    Article Title: A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype Relationships
    Article Snippet: SLiCE reactions were 10 μL and consisted of 100 ng of linearized vector with 15 ng of tile DNA, along with 1× SLiCE buffer and 1× SLiCE extract. .. Reactions were incubated for 60 min at 37°C, then diluted 1:10 in water, and 2.5 μL used to electroporate 50 μL of NEB 10-beta electrocompetent E. coli .

    Article Title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosirupto
    Article Snippet: Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies). .. Cloning and expression of recombinant proteins used various E. coli strains: NovaBlue GigaSinglesTM (EMD Millipore), RosettaTM 2(DE3) SinglesTM (EMD Millipore), NEB 10-beta electrocompetent E. coli (New England Biolabs), and Arctic Express (DE3)RIL E. coli (Agilent Technologies).

    Article Title: VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016
    Article Snippet: The phylogenetic group was determined by PCR [ ], the class I integron was amplified and the purified amplification products were sequenced as described previously [ ]. .. Plasmid DNA was isolated using the NucleoBond Xtra Midi kit (Macherey-Nagel, Dueren, Germany) and the bla VIM-1 -containing plasmid was transferred into electrocompetent E. coli NEB10-beta (New England Biolabs, Frankfurt a.M., Germany). .. The incompatibility (Inc-) group of the plasmid was determined by using the PCR-based replicon typing (PBRT) kit (Diatheva, Fano, Italy).

    Article Title: New CRISPR-Cas systems from uncultivated microbes
    Article Snippet: Putative targets identified from metagenomic sequence analysis or PAM depletion assays were cloned into a pUC19 plasmid. .. 10 ng of target plasmid were transformed into electrocompetent E. coli (NEB Stable) containing the CRISPR loci plasmid. .. CasX.1 was used for the plasmid interference assays under control of native promoters or using a strong heterologous promoter (J23119) for sgRNA and crRNA expression.

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The combined aqueous layers containing the plasmid DNA were concentrated using a spin column (DNA Clean & Concentrator-5, Zymo Research) and eluted with molecular biology grade water (10 μl). .. Electrocompetent E. coli cells (50 μl, β-10 E. coli cells, NEB) were transformed with 5 μl of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser).

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning. .. DNA plasmid purification from bacteria was performed using QIAPrep Spin Miniprep (Qiagen), Zyppy Plasmid Midiprep (Zymo Research) or E.Z.N.A.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Paragraph title: CRISPR plasmid and strain construction ... Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs).

    Article Title: Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
    Article Snippet: 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours. .. 1 μg of ethanol-precipitated ligate was electroporated at 1700V into 25 μl of Electrocompetent NEB 10-beta (New England Biolabs), and the culture was grown in S.O.C. media with shaking at room temperature for 1.5 hours.

    Article Title: Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
    Article Snippet: Electrocompetent NEB 10-beta cells (New England Biolabs) were electroporated with two plasmids. .. The first plasmid expresses Cas9 (inducible by anhydrotetracycline, ATc), the sgRNA (inducible by arabinose), and a spectinomycin resistance gene.

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The vector backbone was digested with AgeI and EcoRI, treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), purified on a 1% agarose gel followed by gel extraction (Qiagen). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Article Title: A platform for functional assessment of large variant libraries in mammalian cells
    Article Snippet: This double-stranded DNA was ligated into the kanamycin-resistant plasmid attB_GPiM upstream of the IRES element using directional cloning with the EcoRI-HF and SacII restriction enzymes (New England Biolabs). .. The ligated product was cleaned with a Zymo clean and concentrator kit (Zymo Research), and electroporated into NEB 10-beta Electrocompetent Escherichia coli (New England Biolabs), yielding an estimated 2 million unique transformants as determined by dilution plating and colony count.

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The full-length cassette was then assembled by combining 15 ng of each fragment and DNA primers P1.For and P3.Rev into a single PCR reaction. .. The PCR-amplified cassette was digested with AscI and BglII restriction enzymes, ligated into the pGDR11 expression vector and transformed into electrocompetent 10-beta E. coli (New England Biolabs Inc., Massachusetts, USA). .. Polymerase variants were grown as a population of E. coli carrying the pGDR11 plasmid encoding the polymerase of interest in Luria–Bertani (LB) broth supplemented with ampicillin (100 μg ml−1 ).

    Article Title: Automated multiplex genome-scale engineering in yeast
    Article Snippet: The CAD strain was constructed previously via integration of an RNAi pathway into the CEN.PK2-1c genome. .. Zymo 5α Z-competent E. coli (Zymo Research, Irvine, CA) and NEB 10β Electrocompetent E. coli (New England Biolabs, Ipswich, MA) were used for plasmid amplification and library construction, respectively. .. S. cerevisiae strains were cultivated in either synthetic complete (SC) dropout medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate and 0.083% amino-acid dropout mix, 0.01% adenine hemisulfate and 2% glucose) or YPAD medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate and 2% glucose).

    Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
    Article Snippet: The concentration of ligated vector was determined by Qubit dsDNA HS Assay (Life Technologies). .. A total of ten electroporations were performed according to the manufacturer’s protocol using 1 ng of ligated vector and 25 ul of DH10beta E. coli Electrocompetent Cells (NEB). .. Each electroporation reaction was then plated onto two 15-cm diameter agar plates containing Luria broth medium (Life Technologies) and 100 μg/ml carbenicillin.

    Electrophoresis:

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The PCR-amplified library was then purified (Qiagen), digested with BbsI (New England Biolabs) at 37 °C overnight, and purified by electrophoresis on a 2% agarose gel and recovered using gel extraction kit (Qiagen). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Agarose Gel Electrophoresis:

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The vector backbone was digested with AgeI and EcoRI, treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), purified on a 1% agarose gel followed by gel extraction (Qiagen). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Produced:

    Article Title: Integrated mechanism for the generation of the 5′ junctions of LINE inserts
    Article Snippet: After a 10-day incubation, HeLa-RC cell clones derived from each G418-resistant colony produced by pLEmH, TK109-17 or Nb2A3-2 were cultured separately until the total number of cells reached ∼1 × 106 cells per clone. .. Ninety percent of the circular DNA was incorporated in Escherichia coli ElectroMAX DH10B Cells (Invitrogen) or NEB 10-beta Electrocompetent E. coli (New England Biolabs) by electroporation with the GENE Pulser Xcell (Bio-Rad) under conditions of 2500 V, 25 mF and 100 V, and the electroporated cells were plated on plates containing kanamycin (70 mg/ml).

    Concentration Assay:

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The custom oligonucleotide library was reconstituted in water to a final concentration of 0.01 pmol/µL and PCR-amplified using Q5 Hot Start Polymerase (New England Biolabs). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

    Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
    Article Snippet: The concentration of ligated vector was determined by Qubit dsDNA HS Assay (Life Technologies). .. A total of ten electroporations were performed according to the manufacturer’s protocol using 1 ng of ligated vector and 25 ul of DH10beta E. coli Electrocompetent Cells (NEB).

    DNA Purification:

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Phusion DNA Polymerase, Taq polymerase, DNA restriction and ligation enzymes were purchased from New England Biolabs (NEB), and DNA purification and gel-extraction were performed using Geneaid kits. .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning.

    Gel Extraction:

    Article Title: Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen
    Article Snippet: Phusion DNA Polymerase, Taq polymerase, DNA restriction and ligation enzymes were purchased from New England Biolabs (NEB), and DNA purification and gel-extraction were performed using Geneaid kits. .. DNA sequences were verified by Genewiz, Inc. E scherichia coli strains DH5α, 10-β electrocompetent (NEB) were used as hosts for molecular cloning.

    Article Title: CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
    Article Snippet: Plasmid 44251 digested with SpeI and ApaI (New England Biolabs) was used as the cloning backbone. .. Digested inserts were gel extracted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific) and ligated (T4 DNA ligase, New England Biolabs) alongside this backbone and transformed into electrocompetent NEB 10-β (New England Biolabs). .. Final constructs were recovered using Zyppy Plasmid Miniprep Kit (Zymo Research Corporation) and confirmed by sequencing (via GENEWIZ) before transformation into chemically competent E. coli MG1655 (ATCC 700926) harboring either dCas9 or dCas9-ω plasmids for gene repression or activation respectively.

    Article Title: SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling
    Article Snippet: The vector backbone was digested with AgeI and EcoRI, treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), purified on a 1% agarose gel followed by gel extraction (Qiagen). .. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of 10-beta electrocompetent cells (New England Biolabs).

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