ecorv hf  (New England Biolabs)


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    Name:
    EcoRV HF
    Description:
    EcoRV HF 20 000 units
    Catalog Number:
    r3195l
    Price:
    249
    Size:
    20 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs ecorv hf
    EcoRV HF
    EcoRV HF 20 000 units
    https://www.bioz.com/result/ecorv hf/product/New England Biolabs
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    ecorv hf - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing"

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00070

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.
    Figure Legend Snippet: (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Techniques Used: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay

    Related Articles

    Amplification:

    Article Title: TRPV6 Variants Interfere with Maternal-Fetal Calcium Transport through the Placenta and Cause Transient Neonatal Hyperparathyroidism
    Article Snippet: .. Amplified fragments were treated with T4 polynucleotide kinase (Takara, Japan), ligated by T4 ligase (ToYoBo, Japan) with pcDNA3.1 (+) (Life Technologies, USA), and pretreated with Eco R V-HF (NEB, USA) and CIP (Takara, Japan). ..

    Ligation:

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe
    Article Snippet: .. Identification of insertion sites by inverse splinkerette PCR Genomic DNA (10 μg) was digested with 80 units of EcoR V-HF (NEB) at 37°C for 16 h, followed by 65°C denaturation for 15 min. A total of 1.5 μg of digested DNA was used in a 500-μl ligation (final DNA concentration = 3 ng/μl) with 2000 units of T4 DNA ligase at 16°C for 16 h. Ligated DNA was subsequently digested with 60 units of Sfi I restriction endonuclease at 50°C for five hours. .. All of the Sfi I-digested DNA was used in a 30-μl ligation reaction that contained 0.33 μM of double-strand Sfi I splinkerette and 800 units of T4 DNA ligase.

    Ethanol Precipitation:

    Article Title: Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions
    Article Snippet: .. After phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation, the recovered plasmids were treated with or without Eco RV-HF (New England Biolabs). .. E. coli strain NEB10β (New England Biolabs) was transformed with the mixture of above plasmids and 0.08 ng of pZeo, which serves as an internal control for transformation efficiency, and then plated onto 1xYT plates with carbenicillin/blasticidin S and 1xYT plates with zeocin, respectively.

    Concentration Assay:

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe
    Article Snippet: .. Identification of insertion sites by inverse splinkerette PCR Genomic DNA (10 μg) was digested with 80 units of EcoR V-HF (NEB) at 37°C for 16 h, followed by 65°C denaturation for 15 min. A total of 1.5 μg of digested DNA was used in a 500-μl ligation (final DNA concentration = 3 ng/μl) with 2000 units of T4 DNA ligase at 16°C for 16 h. Ligated DNA was subsequently digested with 60 units of Sfi I restriction endonuclease at 50°C for five hours. .. All of the Sfi I-digested DNA was used in a 30-μl ligation reaction that contained 0.33 μM of double-strand Sfi I splinkerette and 800 units of T4 DNA ligase.

    Luciferase:

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    Expressing:

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    Polymerase Chain Reaction:

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing
    Article Snippet: .. Three microliters of PCR product was digested with 5 units of EcoRV-HF (New England Biolabs) for 2 h in a standard 10 μl restriction enzyme reaction following the manufacturer's instructions. .. Each 3 μL of digested PCR product was electrophoresed on a 2.0% agarose, 0.5 X TBE gel to determine if genome editing had occurred.

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe
    Article Snippet: .. Identification of insertion sites by inverse splinkerette PCR Genomic DNA (10 μg) was digested with 80 units of EcoR V-HF (NEB) at 37°C for 16 h, followed by 65°C denaturation for 15 min. A total of 1.5 μg of digested DNA was used in a 500-μl ligation (final DNA concentration = 3 ng/μl) with 2000 units of T4 DNA ligase at 16°C for 16 h. Ligated DNA was subsequently digested with 60 units of Sfi I restriction endonuclease at 50°C for five hours. .. All of the Sfi I-digested DNA was used in a 30-μl ligation reaction that contained 0.33 μM of double-strand Sfi I splinkerette and 800 units of T4 DNA ligase.

    Plasmid Preparation:

    Article Title: A transcriptional circuit filters oscillating circadian hormonal inputs to regulate fat cell differentiation
    Article Snippet: .. The pENTR1a backbone vector was digested with EcoR Ι-HF and BamH Ι-HF (NEB), and assembled together with three DNA fragments coding for homology arm 1, Citrine or mKate2, and homology arm 2 using Gibson assembly. .. The homology arm fragments were PCR amplified from OP9 genomic DNA with primers introducing a 15–20 bp overhang used for Gibson assembly.

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

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    New England Biolabs restriction enzyme ecori hf
    Dendrogram of <t>EcoRI-digested</t> plasmids from 27 <t>transconjugants.</t> 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.
    Restriction Enzyme Ecori Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme ecori hf/product/New England Biolabs
    Average 99 stars, based on 16 article reviews
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    Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.

    Journal: PLoS ONE

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae

    doi: 10.1371/journal.pone.0133654

    Figure Lengend Snippet: Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.

    Article Snippet: Transconjugants plasmids were digested with the restriction enzyme EcoRI -HF (New England Biolabs, UK) [ ].

    Techniques: Plasmid Preparation

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.

    Journal: PLoS ONE

    Article Title: Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans

    doi: 10.1371/journal.pone.0026594

    Figure Lengend Snippet: PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.

    Article Snippet: All regions were cut with DraI (NEB) to identify SNPs, except for 2.8 LG.I the DNA was digested EcoRI-HF (NEB).

    Techniques: Derivative Assay, Clone Assay, Positive Control, Laser Capture Microdissection, Staining

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Journal: bioRxiv

    Article Title: Depurination of colibactin-derived interstrand cross-links

    doi: 10.1101/869313

    Figure Lengend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Article Snippet: To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop.

    Techniques: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis