dh5α  (New England Biolabs)


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    Structured Review

    New England Biolabs dh5α
    Dh5α, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dh5α/product/New England Biolabs
    Average 93 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    dh5α - by Bioz Stars, 2020-02
    93/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells
    Article Snippet: .. Escherichia coli K12 and DH5α (New England Biolabs, Germany) were used for cloning purposes, while E. coli DH5α F’ I q ([ F´ proA + B + lacI q ∆(lacZ)M15 zzf::Tn10 (TetR ) /fhuA2∆(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 ]; New England Biolabs, Germany) and E. coli OverExpress C43(DE3) ([F– ompT hsdS B (r B – m B – ) gal dcm (DE3)]; Lucigen Corporation, USA) were employed for protein expression. ..

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol. .. One hundred and two (102) recombinant clones were obtained.

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: This fragment was cloned into the plasmid pfa6a-GFP-kanR through EcoRI and SalI (now named pfa6a-GRX1). .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together).

    Article Title: Function of the Borrelia burgdorferi FtsH Homolog Is Essential for Viability both In Vitro and In Vivo and Independent of HflK/C
    Article Snippet: .. Cloning vectors were propagated using E. coli strain TOP10 (Invitrogen, Carlsbad, CA) or DH5α (New England Biolabs, Ipswich, MA). ..

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    Amplification:

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The GeneMorph II Random Mutagenesis Kit (Agilent) was used to amplify the GRX1 core promoter with random mutations from pfa6a-GRX1 with primers C5 and C6 following the standard protocol of the manufacturer (a total of 50 cycles of amplification was applied). .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together).

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: Bacterial strains and plasmids Genomic DNA from R. cellulolyticum ATCC 35319 (NCBI Reference Sequence: NC_011898.1) served as a template for amplification by PCR of the genes at loci Ccel_1229, Ccel_1231, Ccel_1233, Ccel_1234, Ccel_1235, and Ccel_1240 encoding the mature forms of the putative α-l -arabinofuranosidases. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    Construct:

    Article Title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
    Article Snippet: Plasmid construction Plasmids were propagated in E. coli strain TOP10 (Invitrogen) or DH5α (NEB), grown in LB medium, containing 100 μg/ml ampicillin at 30°C or 37°C. .. The plasmids pHD8 and pHD22 were constructed by homologous recombination in yeast from up to 17 single overlapping PCR fragments.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection. ..

    Incubation:

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection. ..

    Cell Culture:

    Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
    Article Snippet: .. Cell culture E. coli chemically competent cells were either DH5α (NEB) or TOP10 (Invitrogen). .. High-competent electrochemical competent cells MegaX DH10B T1 (Invitrogen) were used for the random library plasmid transformation.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Paragraph title: Cell culturing and plasmid preparation ... The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection.

    Expressing:

    Article Title: Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells
    Article Snippet: .. Escherichia coli K12 and DH5α (New England Biolabs, Germany) were used for cloning purposes, while E. coli DH5α F’ I q ([ F´ proA + B + lacI q ∆(lacZ)M15 zzf::Tn10 (TetR ) /fhuA2∆(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 ]; New England Biolabs, Germany) and E. coli OverExpress C43(DE3) ([F– ompT hsdS B (r B – m B – ) gal dcm (DE3)]; Lucigen Corporation, USA) were employed for protein expression. ..

    Article Title: Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression
    Article Snippet: .. Strains and media NEB turbo electrocompetent E. coli , DH5α (NEB), LW6, and LW7 ( ) strains (Supplementary Table S1) were grown in Luria-Bertani (LB) medium at 37°C for DNA manipulation or expression experiments. .. Media were supplemented with chloramphenicol (34 ug/ml) to maintain the pLSR plasmid and/or tetracycline (2.5, 5, 10 or 20 ug/ml) to select the lsr operon promoter mutant candidates from the library.

    Modification:

    Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
    Article Snippet: Cell culture E. coli chemically competent cells were either DH5α (NEB) or TOP10 (Invitrogen). .. M. pneumoniae M129 (passage 34) was grown in modified Hayflick medium in 150 cm2 flasks and transformed by electroporation as previously described .

    Article Title: Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells
    Article Snippet: Human liver cancer cells (HepG2) were purchased from ATCC (USA) and grown in Dulbecco's modified Eagle's medium GlutaMAX supplemented with 10% fetal bovine serum, 100 U·ml−1 penicillin and 100 µg·ml−1 streptomycin (all from Gibco Lifescience, Thermo Fischer Scientific, USA). .. Escherichia coli K12 and DH5α (New England Biolabs, Germany) were used for cloning purposes, while E. coli DH5α F’ I q ([ F´ proA + B + lacI q ∆(lacZ)M15 zzf::Tn10 (TetR ) /fhuA2∆(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 ]; New England Biolabs, Germany) and E. coli OverExpress C43(DE3) ([F– ompT hsdS B (r B – m B – ) gal dcm (DE3)]; Lucigen Corporation, USA) were employed for protein expression.

    Transformation Assay:

    Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
    Article Snippet: Cell culture E. coli chemically competent cells were either DH5α (NEB) or TOP10 (Invitrogen). .. High-competent electrochemical competent cells MegaX DH10B T1 (Invitrogen) were used for the random library plasmid transformation.

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: .. Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol. .. To isolate colonies for sequencing, cells from the glycerol stock were spread on X-gal/IPTG/ampicillin-LB agar plates.

    Article Title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
    Article Snippet: Plasmid construction Plasmids were propagated in E. coli strain TOP10 (Invitrogen) or DH5α (NEB), grown in LB medium, containing 100 μg/ml ampicillin at 30°C or 37°C. .. E. coli cells were transformed using the CaCl2 [ ] or electroporation method [ ].

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together). .. 14 transformants were isolated and sequenced to estimate mutation frequencies.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection. ..

    Article Title: Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression
    Article Snippet: Strains and media NEB turbo electrocompetent E. coli , DH5α (NEB), LW6, and LW7 ( ) strains (Supplementary Table S1) were grown in Luria-Bertani (LB) medium at 37°C for DNA manipulation or expression experiments. .. Miller assay experiments were performed using 50 ug/ml ampicillin to maintain the LW7 ( ) strain transformed with pLW11 , pPH01 or pPH14 (Supplementary Table S1).

    Electroporation:

    Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
    Article Snippet: Cell culture E. coli chemically competent cells were either DH5α (NEB) or TOP10 (Invitrogen). .. M. pneumoniae M129 (passage 34) was grown in modified Hayflick medium in 150 cm2 flasks and transformed by electroporation as previously described .

    Article Title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
    Article Snippet: Plasmid construction Plasmids were propagated in E. coli strain TOP10 (Invitrogen) or DH5α (NEB), grown in LB medium, containing 100 μg/ml ampicillin at 30°C or 37°C. .. E. coli cells were transformed using the CaCl2 [ ] or electroporation method [ ].

    Introduce:

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: The amplicons were cloned in pET22b(+) (Novagen) at Nde I/Xho I sites or in pET28b(+) (Novagen) at NcoI/XhoI , to introduce six histidine codons at the 3′ extremity of the coding sequence, except, for the gene encoding, the putative α-l -arabinofuranosidases GH4329 -1231 which is cloned in the pGEX-5X-2 vector (Sigma-Aldrich) at BamH I/Xho I, to add a GST-tag (glutathion S-transferase) at the N-terminal extremity of the recombinant protein. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    DNA Sequencing:

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    Polymerase Chain Reaction:

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: The tagged sequences were captured using streptavidin - coated magnetic beads, then PCR-amplified using SuperSNX24 Forward: 5′-GTTTAAGGCCTAGCTAGCAGAATC-3′ and SuperSNX24+4P Reverse: 5′-pGATTCT GCTAGCTAGGCCTTAAACAAAA-3′ adaptator sequences as primers [ ]. .. Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol.

    Article Title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
    Article Snippet: Plasmid construction Plasmids were propagated in E. coli strain TOP10 (Invitrogen) or DH5α (NEB), grown in LB medium, containing 100 μg/ml ampicillin at 30°C or 37°C. .. The plasmids pHD8 and pHD22 were constructed by homologous recombination in yeast from up to 17 single overlapping PCR fragments.

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The purified PCR product was used as primers to amplify the rest of the plasmids by the Phusion DNA polymerase (NEB). .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together).

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: Bacterial strains and plasmids Genomic DNA from R. cellulolyticum ATCC 35319 (NCBI Reference Sequence: NC_011898.1) served as a template for amplification by PCR of the genes at loci Ccel_1229, Ccel_1231, Ccel_1233, Ccel_1234, Ccel_1235, and Ccel_1240 encoding the mature forms of the putative α-l -arabinofuranosidases. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    Recombinant:

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol. .. One hundred and two (102) recombinant clones were obtained.

    Article Title: CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT)
    Article Snippet: Recombinant clostridial strains were selected on 75 µg ml−1 of thiamphenicol or 40 µg ml−1 erythromycin. .. E. coli strains used in the construction of shuttle vectors were DH5α (NEB) or Turbo (NEB), and were selected on chloramphenicol (25 µg ml−1 ) or erythromycin (500 µg ml−1 ).

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used. .. Protein production and purification Recombinant proteins were purified from 700 mL cultures in lysogenic broth medium supplemented with glycerol (0.85%) and the appropriate antibiotic (at the final concentration of 200 µg/mL for ampicillin and 100 µg/mL for kanamycin).

    Cellular Antioxidant Activity Assay:

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: PCR products were subsequently hybridized with three biotinylated microsatellite probes, corresponding to motifs (CAA)8 , (TG)12 and (AG)12 . .. Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol.

    Magnetic Beads:

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: The tagged sequences were captured using streptavidin - coated magnetic beads, then PCR-amplified using SuperSNX24 Forward: 5′-GTTTAAGGCCTAGCTAGCAGAATC-3′ and SuperSNX24+4P Reverse: 5′-pGATTCT GCTAGCTAGGCCTTAAACAAAA-3′ adaptator sequences as primers [ ]. .. Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol.

    Mutagenesis:

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The GeneMorph II Random Mutagenesis Kit (Agilent) was used to amplify the GRX1 core promoter with random mutations from pfa6a-GRX1 with primers C5 and C6 following the standard protocol of the manufacturer (a total of 50 cycles of amplification was applied). .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together).

    Article Title: Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression
    Article Snippet: Strains and media NEB turbo electrocompetent E. coli , DH5α (NEB), LW6, and LW7 ( ) strains (Supplementary Table S1) were grown in Luria-Bertani (LB) medium at 37°C for DNA manipulation or expression experiments. .. Media were supplemented with chloramphenicol (34 ug/ml) to maintain the pLSR plasmid and/or tetracycline (2.5, 5, 10 or 20 ug/ml) to select the lsr operon promoter mutant candidates from the library.

    Isolation:

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together). .. 14 transformants were isolated and sequenced to estimate mutation frequencies.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Plasmids were isolated using either Monarch (NEB) or PureYield (Promega) Plasmid Miniprep. .. The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection.

    Purification:

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together). .. 14 transformants were isolated and sequenced to estimate mutation frequencies.

    Sequencing:

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol. .. To isolate colonies for sequencing, cells from the glycerol stock were spread on X-gal/IPTG/ampicillin-LB agar plates.

    Article Title: New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica
    Article Snippet: Paragraph title: 2.1. Bacterial strains, culture media, chemicals, and sequencing methods ... Escherichia coli C 41 (λDE3) ( ) and DH5α (New England Biolabs) strains were grown at 37 °C in lysogeny broth (LB, Difco).

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The PCR fragment containing the GRX1 promoter (400 bp core sequence) and its flanking sequence (about 300 bp) was amplified from the genomic DNA of the GRX1-GFP-HIS3MX6 strain with primers C3 and C4. .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together).

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: In this case, the DNA sequence encoding the hexa-histidine tail was introduced in the primer 1231pGexrev. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    Article Title: In Streptomyces lividans, acetyl-CoA synthetase activity is controlled by O-serine and Nε-lysine acetylation
    Article Snippet: Paragraph title: Bacterial strains, culture media, chemicals, and sequencing methods ... Escherichia coli C41 (λDE3) ( ) and DH5α (New England Biolabs) strains were grown in lysogeny broth (LB, Difco) at 37°C.

    Plasmid Preparation:

    Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
    Article Snippet: Cell culture E. coli chemically competent cells were either DH5α (NEB) or TOP10 (Invitrogen). .. High-competent electrochemical competent cells MegaX DH10B T1 (Invitrogen) were used for the random library plasmid transformation.

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: .. Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol. .. To isolate colonies for sequencing, cells from the glycerol stock were spread on X-gal/IPTG/ampicillin-LB agar plates.

    Article Title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
    Article Snippet: .. Plasmid construction Plasmids were propagated in E. coli strain TOP10 (Invitrogen) or DH5α (NEB), grown in LB medium, containing 100 μg/ml ampicillin at 30°C or 37°C. .. E. coli cells were transformed using the CaCl2 [ ] or electroporation method [ ].

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: This fragment was cloned into the plasmid pfa6a-GFP-kanR through EcoRI and SalI (now named pfa6a-GRX1). .. The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together).

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Paragraph title: Cell culturing and plasmid preparation ... The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection.

    Article Title: The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action
    Article Snippet: The amplicons were cloned in pET22b(+) (Novagen) at Nde I/Xho I sites or in pET28b(+) (Novagen) at NcoI/XhoI , to introduce six histidine codons at the 3′ extremity of the coding sequence, except, for the gene encoding, the putative α-l -arabinofuranosidases GH4329 -1231 which is cloned in the pGEX-5X-2 vector (Sigma-Aldrich) at BamH I/Xho I, to add a GST-tag (glutathion S-transferase) at the N-terminal extremity of the recombinant protein. .. The BL21(DE3) Escherichia coli strain was used as production strain for all the recombinant proteins, except for GH4329 -1231 for which the DH5α (New England Biolabs) strain was used.

    Article Title: Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression
    Article Snippet: Strains and media NEB turbo electrocompetent E. coli , DH5α (NEB), LW6, and LW7 ( ) strains (Supplementary Table S1) were grown in Luria-Bertani (LB) medium at 37°C for DNA manipulation or expression experiments. .. Media were supplemented with chloramphenicol (34 ug/ml) to maintain the pLSR plasmid and/or tetracycline (2.5, 5, 10 or 20 ug/ml) to select the lsr operon promoter mutant candidates from the library.

    Selection:

    Article Title: Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library
    Article Snippet: Final microsatellite-rich products were inserted into the pGEM-T vector (Promega) and transformed into DH5α (NEB) competent cells according to the standard protocol. .. Screening these clones using T7 and SP6 PCR primers enabled the selection of 62 clones harbouring inserts varying in length between 500 bp and 1300 bp.

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together). .. Then URA3 was replaced by the fragments of pfa6a-GRX1 containing a variant promoter which was cut from the pooled plasmids by EcoRI and SalI through 5-FOA selection (a total of 105 transformants were obtained and pooled together).

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. The Inoue Method [ ] was used to prepare home-made ultra-competent cells of DH5α (NEB) or TOP10 (Thermo Fisher Scientific), which were transformed with the constructs as follows: 5 μl of the DNA solution was incubated with 50 μl of the competent cells on ice for 10 min, followed by a heat shock at 42°C for 40 s and re-cooled on ice for 10 min. SOC medium (400 μl) was added, and after 1 hr incubation at 37°C, 200 rpm, 50 μl of the cell suspension was plated on LB agar plates with antibiotic selection. ..

    Variant Assay:

    Article Title: A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast
    Article Snippet: The final product was digested by DpnI, purified, and then transformed to DH5α (NEB) following the standard protocol of the manufacturer (a total of 105 transformants were obtained and pooled together). .. Then URA3 was replaced by the fragments of pfa6a-GRX1 containing a variant promoter which was cut from the pooled plasmids by EcoRI and SalI through 5-FOA selection (a total of 105 transformants were obtained and pooled together).

    Homologous Recombination:

    Article Title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
    Article Snippet: Plasmid construction Plasmids were propagated in E. coli strain TOP10 (Invitrogen) or DH5α (NEB), grown in LB medium, containing 100 μg/ml ampicillin at 30°C or 37°C. .. The plasmids pHD8 and pHD22 were constructed by homologous recombination in yeast from up to 17 single overlapping PCR fragments.

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  • 92
    New England Biolabs e coli strain dh5α
    The biological parts utilised for combinatorial construction of a lycopene biosynthetic pathway. a The carotenoid lycopene is produced in E. coli through a heterologous biosynthetic pathway composed of three enzymes (CrtE. CrtB and CrtI). The heterologous proteins use FPP, which is a product of MEP pathway, as a precursor. b : Table S1). The pathway library was assembled and expressed in E. coli <t>DH5α</t> to test lycopene titres. The relative effects of the different design factors had on the titres level of lycopene were modelled using JMP. The model identified biological parts which effected titre levels, this can help aid future designing of the lycopene biosynthetic pathway.
    E Coli Strain Dh5α, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 4 article reviews
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    93
    New England Biolabs competent dh5α cells
    Instability of octarepeats during DNA replication in mismatch repair-deficient XL-1 Red cells. (A) Mutant clones from replication of pOct5 in XL-1 Red E.coli cells. Plasmid DNA samples were prepared from XL-1 Red colonies after transformation with pOct5 and re-transformed into <t>DH5α</t> cells. Plasmid DNAs from the resulting DH5α colonies were digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 DH5α colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in XL-1 Red E.coli cells. Same as in (A) except that pOct11b was used. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in XL-1 Red E.coli cells. Shown are plasmid DNAs from two pOct11b-transformed XL-1 Red colonies that produced 2–3 octarepeat bands upon digestion with Sac II and Spe I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence of mixed plasmid DNA species in these colonies. Further transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one of the plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.
    Competent Dh5α Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dh5α isolated pbbr1mcs 5
    Impact of in vitro and in vivo methylation on restriction by NspV. Plasmid DNA was isolated from either E. coli strain <t>DH5α</t> or EAM1 and, where indicated, treated with methylase M.SssI. The plasmid DNA (DH5α-isolated <t>pBBR1MCS-5;</t> DH5α-isolated
    Dh5α Isolated Pbbr1mcs 5, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The biological parts utilised for combinatorial construction of a lycopene biosynthetic pathway. a The carotenoid lycopene is produced in E. coli through a heterologous biosynthetic pathway composed of three enzymes (CrtE. CrtB and CrtI). The heterologous proteins use FPP, which is a product of MEP pathway, as a precursor. b : Table S1). The pathway library was assembled and expressed in E. coli DH5α to test lycopene titres. The relative effects of the different design factors had on the titres level of lycopene were modelled using JMP. The model identified biological parts which effected titre levels, this can help aid future designing of the lycopene biosynthetic pathway.

    Journal: Journal of Biological Engineering

    Article Title: Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway

    doi: 10.1186/s13036-019-0141-z

    Figure Lengend Snippet: The biological parts utilised for combinatorial construction of a lycopene biosynthetic pathway. a The carotenoid lycopene is produced in E. coli through a heterologous biosynthetic pathway composed of three enzymes (CrtE. CrtB and CrtI). The heterologous proteins use FPP, which is a product of MEP pathway, as a precursor. b : Table S1). The pathway library was assembled and expressed in E. coli DH5α to test lycopene titres. The relative effects of the different design factors had on the titres level of lycopene were modelled using JMP. The model identified biological parts which effected titre levels, this can help aid future designing of the lycopene biosynthetic pathway.

    Article Snippet: The E. coli strain DH5α (NEB, USA) was used for all DNA cloning.

    Techniques: Produced

    The biological parts utilised for combinatorial construction of a lycopene biosynthetic pathway. a The carotenoid lycopene is produced in E. coli through a heterologous biosynthetic pathway composed of three enzymes (CrtE. CrtB and CrtI). The heterologous proteins use FPP, which is a product of MEP pathway, as a precursor. b A complete combinatorial library totalling 810 pathway configurations can be designed by varying the promoter and RBS parts and by varying the order of pathway genes and RBS parts. Through the application of statistical modelling by DoE software, the designed library was reduced to 88 representative constructs, A table displaying part information of each construct is displayed in the supplementary information ( Additional file 1 : Table S1). The pathway library was assembled and expressed in E. coli DH5α to test lycopene titres. The relative effects of the different design factors had on the titres level of lycopene were modelled using JMP. The model identified biological parts which effected titre levels, this can help aid future designing of the lycopene biosynthetic pathway.

    Journal: Journal of Biological Engineering

    Article Title: Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway

    doi: 10.1186/s13036-019-0141-z

    Figure Lengend Snippet: The biological parts utilised for combinatorial construction of a lycopene biosynthetic pathway. a The carotenoid lycopene is produced in E. coli through a heterologous biosynthetic pathway composed of three enzymes (CrtE. CrtB and CrtI). The heterologous proteins use FPP, which is a product of MEP pathway, as a precursor. b A complete combinatorial library totalling 810 pathway configurations can be designed by varying the promoter and RBS parts and by varying the order of pathway genes and RBS parts. Through the application of statistical modelling by DoE software, the designed library was reduced to 88 representative constructs, A table displaying part information of each construct is displayed in the supplementary information ( Additional file 1 : Table S1). The pathway library was assembled and expressed in E. coli DH5α to test lycopene titres. The relative effects of the different design factors had on the titres level of lycopene were modelled using JMP. The model identified biological parts which effected titre levels, this can help aid future designing of the lycopene biosynthetic pathway.

    Article Snippet: The E. coli strain DH5α (NEB, USA) was used for all DNA cloning.

    Techniques: Produced, Software, Construct

    Instability of octarepeats during DNA replication in mismatch repair-deficient XL-1 Red cells. (A) Mutant clones from replication of pOct5 in XL-1 Red E.coli cells. Plasmid DNA samples were prepared from XL-1 Red colonies after transformation with pOct5 and re-transformed into DH5α cells. Plasmid DNAs from the resulting DH5α colonies were digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 DH5α colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in XL-1 Red E.coli cells. Same as in (A) except that pOct11b was used. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in XL-1 Red E.coli cells. Shown are plasmid DNAs from two pOct11b-transformed XL-1 Red colonies that produced 2–3 octarepeat bands upon digestion with Sac II and Spe I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence of mixed plasmid DNA species in these colonies. Further transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one of the plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.

    Journal: PLoS ONE

    Article Title: Instability of the Octarepeat Region of the Human Prion Protein Gene

    doi: 10.1371/journal.pone.0026635

    Figure Lengend Snippet: Instability of octarepeats during DNA replication in mismatch repair-deficient XL-1 Red cells. (A) Mutant clones from replication of pOct5 in XL-1 Red E.coli cells. Plasmid DNA samples were prepared from XL-1 Red colonies after transformation with pOct5 and re-transformed into DH5α cells. Plasmid DNAs from the resulting DH5α colonies were digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 DH5α colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in XL-1 Red E.coli cells. Same as in (A) except that pOct11b was used. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in XL-1 Red E.coli cells. Shown are plasmid DNAs from two pOct11b-transformed XL-1 Red colonies that produced 2–3 octarepeat bands upon digestion with Sac II and Spe I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence of mixed plasmid DNA species in these colonies. Further transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one of the plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.

    Article Snippet: The PCR products were treated with the Wizard PCR clean-up kit (Promega, WI, USA), ligated to pGEM-T (for Pwo-amplified products, A-tailing was done first with Taq DNA polymerase in PCR buffer with 0.2 mM dATP) and transformed into competent DH5α cells (New England Biolabs, MA, USA) on LB-agar plates with X-gal.

    Techniques: Mutagenesis, Clone Assay, Plasmid Preparation, Transformation Assay, Agarose Gel Electrophoresis, Produced, Sequencing

    Mutant plasmids from octarepeat replication in DH5α are all head-to-head dimers. (A) Restriction analysis of replication-mutant plasmids with Spe I, Sac II and Sca I. pOct5 or pOct11b were transformed into DH5α. Plasmid DNAs were prepared from two mutant colonies and two control (non-mutant) colonies each for pOct5 and pOct11b, digested with Spe I, Sac II or Sca I, and separated by agarose gel electrophoresis. All mutant colonies appear to contain a minute amount of monomer plasmids. M1, 100-bp DNA ladder; M2, 1-kb DNA ladder. (B) Diagram of the head-to-head plasmid dimers. The top panel depicts the parental plasmid monomer; the bottom panel depicts the dimer where the newly generated monomer unit is highlighted in thicker lines. The boxes denote the octarepeat inserts.

    Journal: PLoS ONE

    Article Title: Instability of the Octarepeat Region of the Human Prion Protein Gene

    doi: 10.1371/journal.pone.0026635

    Figure Lengend Snippet: Mutant plasmids from octarepeat replication in DH5α are all head-to-head dimers. (A) Restriction analysis of replication-mutant plasmids with Spe I, Sac II and Sca I. pOct5 or pOct11b were transformed into DH5α. Plasmid DNAs were prepared from two mutant colonies and two control (non-mutant) colonies each for pOct5 and pOct11b, digested with Spe I, Sac II or Sca I, and separated by agarose gel electrophoresis. All mutant colonies appear to contain a minute amount of monomer plasmids. M1, 100-bp DNA ladder; M2, 1-kb DNA ladder. (B) Diagram of the head-to-head plasmid dimers. The top panel depicts the parental plasmid monomer; the bottom panel depicts the dimer where the newly generated monomer unit is highlighted in thicker lines. The boxes denote the octarepeat inserts.

    Article Snippet: The PCR products were treated with the Wizard PCR clean-up kit (Promega, WI, USA), ligated to pGEM-T (for Pwo-amplified products, A-tailing was done first with Taq DNA polymerase in PCR buffer with 0.2 mM dATP) and transformed into competent DH5α cells (New England Biolabs, MA, USA) on LB-agar plates with X-gal.

    Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Generated

    Schematic diagrams for analysis of octarepeat mutation rate during PCR or replication in E.coli . (A) Measurement of octarepeat mutants resulting from PCR amplification of octarepeats. Cloned PrP ORF sequences (PrP-Oct5 and PrP-Oct11a) were subjected to PCR with primers HP20 and HP306r. The PCR products (depicted in a shaded box), which contained input (green line) and PCR-mutant (red line) octarepeat sequences as well as “paired” molecules (two parallel lines), were cleaned up and ligated to the pGEM-T vector, producing 4 kinds of ligation products: mutant monomer, wild type monomer, wild type dimer, and mutant dimer. The ligation products were transformed into DH5α competent cells, and the resulting colonies were directly examined by PCR with primers HP50f and HP293r. Plasmid DNAs were extracted from colonies containing mutant octarepeats, subjected to restriction analysis with Sac II and Spe I, and sequenced. The number of mutant colonies over the total number of colonies screened was calculated to represent the octarepeat mutation rate during the PCR process. Small oval: individual E.coli cell; big dashed-line oval: E.coli colony. (B) Measurement of octarepeat mutants resulting from DNA replication in E.coli . pOct5 or pOct11b was used to transform competent DH5α or XL-1 Red E.coli cells, and plasmid DNA sample prepared from a single colony (depicted in a shaded box) was used to transform competent DH5α cells. The mutant colonies (containing only mutant plasmid, red dashed-line oval) and new mutant colonies [containing some cells with the NEW replication-mutant octarepeat insert (light purple), light blue dashed-line oval] were screened out as in (A). The number of mutant colonies (excluding the new mutant colonies) over the total number of colonies examined should reflect the octarepeat mutation rate during the 1 st round of plasmid replication and cell division in E.coli (DH5α or XL-1 Red).

    Journal: PLoS ONE

    Article Title: Instability of the Octarepeat Region of the Human Prion Protein Gene

    doi: 10.1371/journal.pone.0026635

    Figure Lengend Snippet: Schematic diagrams for analysis of octarepeat mutation rate during PCR or replication in E.coli . (A) Measurement of octarepeat mutants resulting from PCR amplification of octarepeats. Cloned PrP ORF sequences (PrP-Oct5 and PrP-Oct11a) were subjected to PCR with primers HP20 and HP306r. The PCR products (depicted in a shaded box), which contained input (green line) and PCR-mutant (red line) octarepeat sequences as well as “paired” molecules (two parallel lines), were cleaned up and ligated to the pGEM-T vector, producing 4 kinds of ligation products: mutant monomer, wild type monomer, wild type dimer, and mutant dimer. The ligation products were transformed into DH5α competent cells, and the resulting colonies were directly examined by PCR with primers HP50f and HP293r. Plasmid DNAs were extracted from colonies containing mutant octarepeats, subjected to restriction analysis with Sac II and Spe I, and sequenced. The number of mutant colonies over the total number of colonies screened was calculated to represent the octarepeat mutation rate during the PCR process. Small oval: individual E.coli cell; big dashed-line oval: E.coli colony. (B) Measurement of octarepeat mutants resulting from DNA replication in E.coli . pOct5 or pOct11b was used to transform competent DH5α or XL-1 Red E.coli cells, and plasmid DNA sample prepared from a single colony (depicted in a shaded box) was used to transform competent DH5α cells. The mutant colonies (containing only mutant plasmid, red dashed-line oval) and new mutant colonies [containing some cells with the NEW replication-mutant octarepeat insert (light purple), light blue dashed-line oval] were screened out as in (A). The number of mutant colonies (excluding the new mutant colonies) over the total number of colonies examined should reflect the octarepeat mutation rate during the 1 st round of plasmid replication and cell division in E.coli (DH5α or XL-1 Red).

    Article Snippet: The PCR products were treated with the Wizard PCR clean-up kit (Promega, WI, USA), ligated to pGEM-T (for Pwo-amplified products, A-tailing was done first with Taq DNA polymerase in PCR buffer with 0.2 mM dATP) and transformed into competent DH5α cells (New England Biolabs, MA, USA) on LB-agar plates with X-gal.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Ligation, Transformation Assay

    Instability of octarepeats during DNA replication in DH5α cells. (A) Mutant clones from replication of pOct5 in DH5α cells. pOct5 was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 3 colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in DH5α cells. pOct11b was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in DH5α cells. Shown are plasmid DNAs from two pOct11b-transformed DH5α colonies that produced 2–3 octarepeat bands upon digestion with Sac II and Spe I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence in these colonies of mixed plasmid DNA species where each species produced one of the octarepeat bands. Re-transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.

    Journal: PLoS ONE

    Article Title: Instability of the Octarepeat Region of the Human Prion Protein Gene

    doi: 10.1371/journal.pone.0026635

    Figure Lengend Snippet: Instability of octarepeats during DNA replication in DH5α cells. (A) Mutant clones from replication of pOct5 in DH5α cells. pOct5 was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 3 colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in DH5α cells. pOct11b was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with Sac II and Spe I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in DH5α cells. Shown are plasmid DNAs from two pOct11b-transformed DH5α colonies that produced 2–3 octarepeat bands upon digestion with Sac II and Spe I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence in these colonies of mixed plasmid DNA species where each species produced one of the octarepeat bands. Re-transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.

    Article Snippet: The PCR products were treated with the Wizard PCR clean-up kit (Promega, WI, USA), ligated to pGEM-T (for Pwo-amplified products, A-tailing was done first with Taq DNA polymerase in PCR buffer with 0.2 mM dATP) and transformed into competent DH5α cells (New England Biolabs, MA, USA) on LB-agar plates with X-gal.

    Techniques: Mutagenesis, Clone Assay, Transformation Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Sequencing

    Impact of in vitro and in vivo methylation on restriction by NspV. Plasmid DNA was isolated from either E. coli strain DH5α or EAM1 and, where indicated, treated with methylase M.SssI. The plasmid DNA (DH5α-isolated pBBR1MCS-5; DH5α-isolated

    Journal: Applied and Environmental Microbiology

    Article Title: Species-Specific Type II Restriction-Modification System of Xylella fastidiosa Temecula1 ▿

    doi: 10.1128/AEM.03034-09

    Figure Lengend Snippet: Impact of in vitro and in vivo methylation on restriction by NspV. Plasmid DNA was isolated from either E. coli strain DH5α or EAM1 and, where indicated, treated with methylase M.SssI. The plasmid DNA (DH5α-isolated pBBR1MCS-5; DH5α-isolated

    Article Snippet: To determine if prior methylation would increase X. fastidiosa transformation efficiency, DH5α-isolated pBBR1MCS-5 was treated with M.SssI (New England Biolabs), which methylates DNA at the cytosine residue within its dinucleotide recognition sequence 5′-CG-3′ ( ).

    Techniques: In Vitro, In Vivo, Methylation, Plasmid Preparation, Isolation