coli strains dh5α  (New England Biolabs)


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    Name:
    NEB 5 alpha Competent E coli High Efficiency
    Description:
    NEB 5 alpha Competent E coli High Efficiency 20x0 05 ml
    Catalog Number:
    c2987h
    Price:
    197
    Size:
    1 ml
    Category:
    Competent Bacteria
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    New England Biolabs coli strains dh5α
    NEB 5 alpha Competent E coli High Efficiency
    NEB 5 alpha Competent E coli High Efficiency 20x0 05 ml
    https://www.bioz.com/result/coli strains dh5α/product/New England Biolabs
    Average 95 stars, based on 606 article reviews
    Price from $9.99 to $1999.99
    coli strains dh5α - by Bioz Stars, 2020-02
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    Images

    1) Product Images from "FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli"

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129547

    Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Plasmid Preparation, Transformation Assay, Cell Culture, Incubation

    Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.
    Figure Legend Snippet: Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.

    Techniques Used: Transformation Assay, Incubation, Selection

    Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Transformation Assay

    Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Electroporation Bacterial Transformation, Transformation Assay, Purification, Incubation, Selection

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    Clone Assay:

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    Amplification:

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    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
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    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
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    Reporter Assay:

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    Positive Control:

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    Synthesized:

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    Construct:

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    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
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    Luciferase:

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
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    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
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    Activity Assay:

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: The 6OSE2 Luc plasmid to assess Runx2 activity was amplified as previously described. .. Briefly, 6OSE2 plasmid was transformed into competent DH5-α (Douglas Hanahan 5 alpha) Escherichia coli according to manufacturer’s instructions (C2987H; New England Biolabs, Ipswich, MA).

    Expressing:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
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    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
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    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture. .. 2.2 Expression of hsEH FL in HEK293-F cells A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific).

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture. .. A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific).

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
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    Transformation Assay:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit. .. The recombinant fusion protein- SUMO-MyD88-TIR was expressed in E. coli BL21 C43 (DE3) cells (Lucigen, WI, USA).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: .. Briefly, 6OSE2 plasmid was transformed into competent DH5-α (Douglas Hanahan 5 alpha) Escherichia coli according to manufacturer’s instructions (C2987H; New England Biolabs, Ipswich, MA). .. Plasmid DNA was extracted using plasmid Maxiprep kit (K210026; Invitrogen).

    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
    Article Snippet: .. The pETBlue-2 construct transformed into DH5-alpha E. coli chemically-competent cells ( ) (New England BioLabs Inc., USA Cat No .C2987I) by heat shock and DH5-alpha bacteria were plated on LB agar plates with 100 µg/mL ampicillin. .. The plasmid constructs were transformed into Tuner ™ (DE3) pLacI E. coli chemically-competent cells ( ) (Novagen, USA Cat No .70623) by heat shock and the bacteria were plated on LB agar containing 50µg/ml ampicillin and 20µg/ml chloramphenicol.

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: .. Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions. .. Transfection was done by ViaFectTM Transfection Reagent (E4981, Promega) according to manufacturer instructions with medium to final volume ratio of 4:1.

    Article Title: Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries
    Article Snippet: .. The library was then transformed into C2987 competent cells (NEB) in batch and plated. ..

    Article Title: CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course
    Article Snippet: .. The pCAS plasmid with the guide RNA sequence was then transformed in to DH5α cells (New England Biolabs NEB#C2987H), followed by a plasmid mini prep (Omega EZNA kit #D6942–0) from a single colony. .. The gap repair DNA fragments for ADE2 and STE12 ( ) were commercially synthesized and cloned into the EcoRV site of pUC57 by Genscript.

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Approximately twenty bacteria colonies from multiple sclerosis and normal control subjects, or ten colonies from 293t cells (positive control) and the synthetic 1,738 bp PCR product that covers SHP-1 promoter 2 (negative control), were picked for DNA bisulfite sequencing.

    Article Title: Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts
    Article Snippet: .. DDR1b transfection Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E. coli according to the manufacturer's instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to the manufacturer's instructions (K210026; Invitrogen).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: .. Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E coli according to manufacturer’s instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to manufacturer’s instructions (K210026; Invitrogen).

    Transfection:

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: Paragraph title: 6OSE2 Luc Plasmid Transfection and Luciferase Assay ... Briefly, 6OSE2 plasmid was transformed into competent DH5-α (Douglas Hanahan 5 alpha) Escherichia coli according to manufacturer’s instructions (C2987H; New England Biolabs, Ipswich, MA).

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions. .. Transfection was done by ViaFectTM Transfection Reagent (E4981, Promega) according to manufacturer instructions with medium to final volume ratio of 4:1.

    Article Title: Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts
    Article Snippet: .. DDR1b transfection Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E. coli according to the manufacturer's instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to the manufacturer's instructions (K210026; Invitrogen).

    Ligation:

    Article Title: Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
    Article Snippet: .. pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (Addgene plasmid ID#42335) Oligos for gRNA construction (see ) NEB® 5-alpha Competent E. coli (High Efficiency) (New England Biolabs, cat. no. C2987H) QIAquick gel extraction kit (QIAGEN, cat. no. 28704) QIAprep spin miniprep kit (QIAGEN, cat. no. 27106) SYBR Safe DNA stain, 10,000× (Life Technologies, cat. no. ) FastDigest BbsI (BpiI) (Thermo Scientific, cat. no. FD1014) Roche Rapid Ligation Kit (Roche, cat. 11635379001) NaCl (Merck, cat. S7653) Peptone special (Merck, cat. 68971) Yeast (Merck, cat. 51475) Agar (merck, cat. A5306) Ampicillin (Sigma, cat. no. A9518) Agarose (Sigma, cat. no. A9539) 10X TBE (ThermoFisher, cat. AM9864) Agarose (Merck, cat. A9539) Gene™Ruler DNA Ladder Mix (Thermo Scientific, cat. no.SM0331) Loading Dye (6x) (Thermo Scientific, cat. no. R0611) .. HotStar HighFidelity PCR kit (QIAGEN, cat. no. 202605) Oligos T7 Endonuclease I (New England BioLabs, cat. no.M0302S) NEB buffer 2 (New England BioLabs, cat. no. B7002S)

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: Mutant and Wildtype fragment DNAs were inserted into pNL3.1 vector by Quick Ligation Protocol (M2200, New England Biolabs) using NheI-HF (R3131S, New England Biolabs) and HindII-HF (R3104S, New England Biolabs) according to manufacturer instructions (see Supplementary Table for fragment sequences). .. Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions.

    Cell Culture:

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E coli according to manufacturer’s instructions (C2987H; New England Biolabs). .. Cells were cultured in normal media for 24 hours, followed by protein isolation and analysis by immunoblot as described above.

    Generated:

    Article Title: Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries
    Article Snippet: Random peptide sequences were generated using NNB codons in a 60-base nucleotide segment to produce 20 amino acid long peptides. .. The library was then transformed into C2987 competent cells (NEB) in batch and plated.

    Polymerase Chain Reaction:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: DNA was amplified with the Ready-mix PCR kit (Sigma), and ligated (New England BioLabs, MA, USA) with the restriction enzymes BamH and Xhol (TaKaRa, Kusatsu, Shiga Prefecture, Japan) to pETM-11 SUMO3GFP fusion vector for protein expression in E. coli, a gift from the EMBL protein expression and purification core facility. .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit.

    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
    Article Snippet: To clone full length S. pseudintermedius spsQ and spsQ -M, the PCR products were digested with NotI and BamHI , then ligated into pETBlue-2, an expression vector with C-terminal HSV•Tag® and His•Tag® sequences (Novagen, USA Cat No .70674). .. The pETBlue-2 construct transformed into DH5-alpha E. coli chemically-competent cells ( ) (New England BioLabs Inc., USA Cat No .C2987I) by heat shock and DH5-alpha bacteria were plated on LB agar plates with 100 µg/mL ampicillin.

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: The hsEH FL cDNA was PCR-amplified using primers designed to facilitate directional cloning , as detailed in the kit instructions, generating a blunt-end PCR product that was mixed using a molar ratio of 1:1 of insert:TOPO® vector. .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture.

    Article Title: CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course
    Article Snippet: The gRNA target sequence was inserted into the pCAS plasmid [Addgene plasmid # 60847] through PCR amplification. .. The pCAS plasmid with the guide RNA sequence was then transformed in to DH5α cells (New England Biolabs NEB#C2987H), followed by a plasmid mini prep (Omega EZNA kit #D6942–0) from a single colony.

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Approximately twenty bacteria colonies from multiple sclerosis and normal control subjects, or ten colonies from 293t cells (positive control) and the synthetic 1,738 bp PCR product that covers SHP-1 promoter 2 (negative control), were picked for DNA bisulfite sequencing.

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: The hsEH FL cDNA was PCR-amplified using primers designed to facilitate directional cloning , as detailed in the kit instructions, generating a blunt-end PCR product that was mixed using a molar ratio of 1:1 of insert:TOPO® vector. .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture.

    Recombinant:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: Paragraph title: Recombinant MyD88 TIR Protein ... Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit.

    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
    Article Snippet: Paragraph title: Cloning, expression, and purification of recombinant wild-type and non-toxigenic S. pseudintermedius SpsQ ... The pETBlue-2 construct transformed into DH5-alpha E. coli chemically-competent cells ( ) (New England BioLabs Inc., USA Cat No .C2987I) by heat shock and DH5-alpha bacteria were plated on LB agar plates with 100 µg/mL ampicillin.

    Staining:

    Article Title: Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
    Article Snippet: .. pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (Addgene plasmid ID#42335) Oligos for gRNA construction (see ) NEB® 5-alpha Competent E. coli (High Efficiency) (New England Biolabs, cat. no. C2987H) QIAquick gel extraction kit (QIAGEN, cat. no. 28704) QIAprep spin miniprep kit (QIAGEN, cat. no. 27106) SYBR Safe DNA stain, 10,000× (Life Technologies, cat. no. ) FastDigest BbsI (BpiI) (Thermo Scientific, cat. no. FD1014) Roche Rapid Ligation Kit (Roche, cat. 11635379001) NaCl (Merck, cat. S7653) Peptone special (Merck, cat. 68971) Yeast (Merck, cat. 51475) Agar (merck, cat. A5306) Ampicillin (Sigma, cat. no. A9518) Agarose (Sigma, cat. no. A9539) 10X TBE (ThermoFisher, cat. AM9864) Agarose (Merck, cat. A9539) Gene™Ruler DNA Ladder Mix (Thermo Scientific, cat. no.SM0331) Loading Dye (6x) (Thermo Scientific, cat. no. R0611) .. HotStar HighFidelity PCR kit (QIAGEN, cat. no. 202605) Oligos T7 Endonuclease I (New England BioLabs, cat. no.M0302S) NEB buffer 2 (New England BioLabs, cat. no. B7002S)

    DNA Extraction:

    Article Title: Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts
    Article Snippet: DDR1b transfection Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E. coli according to the manufacturer's instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to the manufacturer's instructions (K210026; Invitrogen).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E coli according to manufacturer’s instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to manufacturer’s instructions (K210026; Invitrogen).

    Methylation:

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. BiQ analyzer software ( ) was used for methylation analysis of each clone.

    Mutagenesis:

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: Mutant and Wildtype fragment DNAs were inserted into pNL3.1 vector by Quick Ligation Protocol (M2200, New England Biolabs) using NheI-HF (R3131S, New England Biolabs) and HindII-HF (R3104S, New England Biolabs) according to manufacturer instructions (see Supplementary Table for fragment sequences). .. Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions.

    Isolation:

    Article Title: Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries
    Article Snippet: The library was then transformed into C2987 competent cells (NEB) in batch and plated. .. Plasmid DNA was isolated from the library and re-transformed into the E. coli W3110 strain at 3 to 5 times coverage.

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Plasmid DNA was isolated with Fast Plasmid mini Kit (5 prime, Cat. No.2300010) and was commercially sequenced (Genewiz, South Plainfield, NJ).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E coli according to manufacturer’s instructions (C2987H; New England Biolabs). .. Cells were cultured in normal media for 24 hours, followed by protein isolation and analysis by immunoblot as described above.

    Subcloning:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: .. Protein constructs, expression, and purification DH5α cells (New England Biolabs (NEB): C2987I) were used for all subcloning steps. .. Rosseta2 cells (Fisher: 71-403-4) were used to express proteins for purification.

    Purification:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit. .. The recombinant fusion protein- SUMO-MyD88-TIR was expressed in E. coli BL21 C43 (DE3) cells (Lucigen, WI, USA).

    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
    Article Snippet: Paragraph title: Cloning, expression, and purification of recombinant wild-type and non-toxigenic S. pseudintermedius SpsQ ... The pETBlue-2 construct transformed into DH5-alpha E. coli chemically-competent cells ( ) (New England BioLabs Inc., USA Cat No .C2987I) by heat shock and DH5-alpha bacteria were plated on LB agar plates with 100 µg/mL ampicillin.

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: .. Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions. .. Transfection was done by ViaFectTM Transfection Reagent (E4981, Promega) according to manufacturer instructions with medium to final volume ratio of 4:1.

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture. .. 2.2 Expression of hsEH FL in HEK293-F cells A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific).

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Approximately twenty bacteria colonies from multiple sclerosis and normal control subjects, or ten colonies from 293t cells (positive control) and the synthetic 1,738 bp PCR product that covers SHP-1 promoter 2 (negative control), were picked for DNA bisulfite sequencing.

    Article Title: Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts
    Article Snippet: DDR1b transfection Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E. coli according to the manufacturer's instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to the manufacturer's instructions (K210026; Invitrogen).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E coli according to manufacturer’s instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to manufacturer’s instructions (K210026; Invitrogen).

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture. .. A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific).

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: .. Protein constructs, expression, and purification DH5α cells (New England Biolabs (NEB): C2987I) were used for all subcloning steps. .. Rosseta2 cells (Fisher: 71-403-4) were used to express proteins for purification.

    Sequencing:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit. .. The recombinant fusion protein- SUMO-MyD88-TIR was expressed in E. coli BL21 C43 (DE3) cells (Lucigen, WI, USA).

    Article Title: Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries
    Article Snippet: Random sequences were cloned into the KpnI and SalI sites using primers with homology to the tether sequence on the reverse primer. .. The library was then transformed into C2987 competent cells (NEB) in batch and plated.

    Article Title: CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course
    Article Snippet: .. The pCAS plasmid with the guide RNA sequence was then transformed in to DH5α cells (New England Biolabs NEB#C2987H), followed by a plasmid mini prep (Omega EZNA kit #D6942–0) from a single colony. .. The gap repair DNA fragments for ADE2 and STE12 ( ) were commercially synthesized and cloned into the EcoRV site of pUC57 by Genscript.

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: The sequence and the position of each primer pair are listed in and , respectively. .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987).

    CRISPR:

    Article Title: CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course
    Article Snippet: Forward and reverse primers were designed based on results from the CRISPR direct online analysis ( ). .. The pCAS plasmid with the guide RNA sequence was then transformed in to DH5α cells (New England Biolabs NEB#C2987H), followed by a plasmid mini prep (Omega EZNA kit #D6942–0) from a single colony.

    IA:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: A synthetic DNA fragment corresponding to the cDNA encoding human MyD88 TIR domain residues 150–296 with BamH/Xhol restriction enzymes was from IDT (Integrated DNA technologies, IA, USA). .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit.

    Plasmid Preparation:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit. .. The recombinant fusion protein- SUMO-MyD88-TIR was expressed in E. coli BL21 C43 (DE3) cells (Lucigen, WI, USA).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: .. Briefly, 6OSE2 plasmid was transformed into competent DH5-α (Douglas Hanahan 5 alpha) Escherichia coli according to manufacturer’s instructions (C2987H; New England Biolabs, Ipswich, MA). .. Plasmid DNA was extracted using plasmid Maxiprep kit (K210026; Invitrogen).

    Article Title: Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
    Article Snippet: .. pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (Addgene plasmid ID#42335) Oligos for gRNA construction (see ) NEB® 5-alpha Competent E. coli (High Efficiency) (New England Biolabs, cat. no. C2987H) QIAquick gel extraction kit (QIAGEN, cat. no. 28704) QIAprep spin miniprep kit (QIAGEN, cat. no. 27106) SYBR Safe DNA stain, 10,000× (Life Technologies, cat. no. ) FastDigest BbsI (BpiI) (Thermo Scientific, cat. no. FD1014) Roche Rapid Ligation Kit (Roche, cat. 11635379001) NaCl (Merck, cat. S7653) Peptone special (Merck, cat. 68971) Yeast (Merck, cat. 51475) Agar (merck, cat. A5306) Ampicillin (Sigma, cat. no. A9518) Agarose (Sigma, cat. no. A9539) 10X TBE (ThermoFisher, cat. AM9864) Agarose (Merck, cat. A9539) Gene™Ruler DNA Ladder Mix (Thermo Scientific, cat. no.SM0331) Loading Dye (6x) (Thermo Scientific, cat. no. R0611) .. HotStar HighFidelity PCR kit (QIAGEN, cat. no. 202605) Oligos T7 Endonuclease I (New England BioLabs, cat. no.M0302S) NEB buffer 2 (New England BioLabs, cat. no. B7002S)

    Article Title: Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius
    Article Snippet: To clone full length S. pseudintermedius spsQ and spsQ -M, the PCR products were digested with NotI and BamHI , then ligated into pETBlue-2, an expression vector with C-terminal HSV•Tag® and His•Tag® sequences (Novagen, USA Cat No .70674). .. The pETBlue-2 construct transformed into DH5-alpha E. coli chemically-competent cells ( ) (New England BioLabs Inc., USA Cat No .C2987I) by heat shock and DH5-alpha bacteria were plated on LB agar plates with 100 µg/mL ampicillin.

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: .. Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions. .. Transfection was done by ViaFectTM Transfection Reagent (E4981, Promega) according to manufacturer instructions with medium to final volume ratio of 4:1.

    Article Title: Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries
    Article Snippet: The surface display system was constructed on the broad host plasmid pMMB67EH. .. The library was then transformed into C2987 competent cells (NEB) in batch and plated.

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture. .. 2.2 Expression of hsEH FL in HEK293-F cells A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific).

    Article Title: CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course
    Article Snippet: .. The pCAS plasmid with the guide RNA sequence was then transformed in to DH5α cells (New England Biolabs NEB#C2987H), followed by a plasmid mini prep (Omega EZNA kit #D6942–0) from a single colony. .. The gap repair DNA fragments for ADE2 and STE12 ( ) were commercially synthesized and cloned into the EcoRV site of pUC57 by Genscript.

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Approximately twenty bacteria colonies from multiple sclerosis and normal control subjects, or ten colonies from 293t cells (positive control) and the synthetic 1,738 bp PCR product that covers SHP-1 promoter 2 (negative control), were picked for DNA bisulfite sequencing.

    Article Title: Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts
    Article Snippet: .. DDR1b transfection Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E. coli according to the manufacturer's instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to the manufacturer's instructions (K210026; Invitrogen).

    Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
    Article Snippet: .. Plasmid containing full-length DDR1b isoform (a gift from the late Dr Wolfgang Vogel) was transformed into competent DH5-α E coli according to manufacturer’s instructions (C2987H; New England Biolabs). .. Plasmid purification was performed using Maxi Prep DNA isolation kit according to manufacturer’s instructions (K210026; Invitrogen).

    Article Title: Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain
    Article Snippet: .. The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture. .. A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific).

    Software:

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. BiQ analyzer software ( ) was used for methylation analysis of each clone.

    Negative Control:

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Approximately twenty bacteria colonies from multiple sclerosis and normal control subjects, or ten colonies from 293t cells (positive control) and the synthetic 1,738 bp PCR product that covers SHP-1 promoter 2 (negative control), were picked for DNA bisulfite sequencing.

    Agarose Gel Electrophoresis:

    Article Title: CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course
    Article Snippet: The pCAS plasmid with the guide RNA sequence was then transformed in to DH5α cells (New England Biolabs NEB#C2987H), followed by a plasmid mini prep (Omega EZNA kit #D6942–0) from a single colony. .. Gap repair fragments for ADE2 and STE12 were confirmed by Genscript and verified by PCR and agarose gel electrophoresis.

    DNA Methylation Assay:

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: To analyze the DNA methylation pattern at the SHP-1 promoter 2, three pairs of PCR primers were used as indicated in the text to amplify promoter 2 sequences: Bis-F (CG2)/Bis-R (CG23) pair covers CpG 2 to 23 of SHP-1 promoter 2, Bis-F (CG13)/Bis-R (CG25) pair ( ) covers CpG 13 to 25, and Bis-F (CG2)/Bis-R (CG25) pair covers CpG 2 to 25. .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987).

    Gel Extraction:

    Article Title: Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88
    Article Snippet: .. Plasmids were gel purified with PureLink Quick Gel Extraction kit (Invitrogen, San Diego, CA, USA), sequence verified, and transformed to (C2987) NEB Dh5a competent cells (New England BioLabs) and amplified with a QIAGEN plasmid midiprep kit. .. The recombinant fusion protein- SUMO-MyD88-TIR was expressed in E. coli BL21 C43 (DE3) cells (Lucigen, WI, USA).

    Article Title: Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
    Article Snippet: .. pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (Addgene plasmid ID#42335) Oligos for gRNA construction (see ) NEB® 5-alpha Competent E. coli (High Efficiency) (New England Biolabs, cat. no. C2987H) QIAquick gel extraction kit (QIAGEN, cat. no. 28704) QIAprep spin miniprep kit (QIAGEN, cat. no. 27106) SYBR Safe DNA stain, 10,000× (Life Technologies, cat. no. ) FastDigest BbsI (BpiI) (Thermo Scientific, cat. no. FD1014) Roche Rapid Ligation Kit (Roche, cat. 11635379001) NaCl (Merck, cat. S7653) Peptone special (Merck, cat. 68971) Yeast (Merck, cat. 51475) Agar (merck, cat. A5306) Ampicillin (Sigma, cat. no. A9518) Agarose (Sigma, cat. no. A9539) 10X TBE (ThermoFisher, cat. AM9864) Agarose (Merck, cat. A9539) Gene™Ruler DNA Ladder Mix (Thermo Scientific, cat. no.SM0331) Loading Dye (6x) (Thermo Scientific, cat. no. R0611) .. HotStar HighFidelity PCR kit (QIAGEN, cat. no. 202605) Oligos T7 Endonuclease I (New England BioLabs, cat. no.M0302S) NEB buffer 2 (New England BioLabs, cat. no. B7002S)

    Article Title: Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects
    Article Snippet: .. PCR products were gel purified using QIAEX II Gel Extraction kit (Qiagen, Cat. No.20021), cloned into pGEM-T vector (Promega, Cat. No.A3600), transformed into NEB 5-alpha competent E. coli (High Efficiency) (NEB, Cat. No.C2987). .. Approximately twenty bacteria colonies from multiple sclerosis and normal control subjects, or ten colonies from 293t cells (positive control) and the synthetic 1,738 bp PCR product that covers SHP-1 promoter 2 (negative control), were picked for DNA bisulfite sequencing.

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    New England Biolabs coli strains dh5α
    Plasmid DNA yield. E . coli <t>DH5α</t> and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Coli Strains Dh5α, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Journal: PLoS ONE

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    doi: 10.1371/journal.pone.0129547

    Figure Lengend Snippet: Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Article Snippet: Bacterial strains and culture conditions The E . coli strains DH5α (NEB #C2987) and JM109 (NEB #E4107) were used for cloning whereas BL21(DE3) (NEB # C2527) along with the other two E . coli strains was used for subsequent experiments.

    Techniques: Plasmid Preparation, Transformation Assay, Cell Culture, Incubation

    Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.

    Journal: PLoS ONE

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    doi: 10.1371/journal.pone.0129547

    Figure Lengend Snippet: Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.

    Article Snippet: Bacterial strains and culture conditions The E . coli strains DH5α (NEB #C2987) and JM109 (NEB #E4107) were used for cloning whereas BL21(DE3) (NEB # C2527) along with the other two E . coli strains was used for subsequent experiments.

    Techniques: Transformation Assay, Incubation, Selection

    Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Journal: PLoS ONE

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    doi: 10.1371/journal.pone.0129547

    Figure Lengend Snippet: Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Article Snippet: Bacterial strains and culture conditions The E . coli strains DH5α (NEB #C2987) and JM109 (NEB #E4107) were used for cloning whereas BL21(DE3) (NEB # C2527) along with the other two E . coli strains was used for subsequent experiments.

    Techniques: Transformation Assay

    Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Journal: PLoS ONE

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    doi: 10.1371/journal.pone.0129547

    Figure Lengend Snippet: Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Article Snippet: Bacterial strains and culture conditions The E . coli strains DH5α (NEB #C2987) and JM109 (NEB #E4107) were used for cloning whereas BL21(DE3) (NEB # C2527) along with the other two E . coli strains was used for subsequent experiments.

    Techniques: Electroporation Bacterial Transformation, Transformation Assay, Purification, Incubation, Selection

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Article Snippet: As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg).

    Techniques: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Article Snippet: As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg).

    Techniques: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Article Snippet: As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg).

    Techniques: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

    Article Snippet: As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg).

    Techniques: Generated, Plasmid Preparation, Chloramphenicol Acetyltransferase Assay, Clone Assay, Polymerase Chain Reaction, Expressing

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Article Snippet: As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg).

    Techniques: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay