dpni enzyme New England Biolabs Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs m774a dpni enzyme new england biolabs
    Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of <t>DNA</t> replication measured by qPCR are reported in pg of <t>DpnI-resistant</t> plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.
    M774a Dpni Enzyme New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 950 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m774a dpni enzyme new england biolabs/product/New England Biolabs
    Average 99 stars, based on 950 article reviews
    Price from $9.99 to $1999.99
    m774a dpni enzyme new england biolabs - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs restriction endonuclease dpni
    Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of <t>DNA</t> replication measured by qPCR are reported in pg of <t>DpnI-resistant</t> plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.
    Restriction Endonuclease Dpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonuclease dpni/product/New England Biolabs
    Average 99 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
    restriction endonuclease dpni - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs rsai
    Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of <t>DNA</t> replication measured by qPCR are reported in pg of <t>DpnI-resistant</t> plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.
    Rsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rsai/product/New England Biolabs
    Average 99 stars, based on 1074 article reviews
    Price from $9.99 to $1999.99
    rsai - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of DNA replication measured by qPCR are reported in pg of DpnI-resistant plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of DNA replication measured by qPCR are reported in pg of DpnI-resistant plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.

    Article Snippet: To measure the amount of replicated pFLORI31 or pFLORI40, 25 μl of total genomic DNA was digested with 10 units of DpnI (New England Biolabs) for 16 hrs in a final volume of 30 μl.

    Techniques: Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Transfection

    Real-time PCR analysis of viral DNA synthesis in Sf9 cells. Total DNA was isolated from Sf9 cells transfected with vAc ac76-KO-PH-GFP or vAc-GP64-KO at selected time points, digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and assayed

    Journal: Journal of Virology

    Article Title: Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation ▿

    doi: 10.1128/JVI.02103-09

    Figure Lengend Snippet: Real-time PCR analysis of viral DNA synthesis in Sf9 cells. Total DNA was isolated from Sf9 cells transfected with vAc ac76-KO-PH-GFP or vAc-GP64-KO at selected time points, digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and assayed

    Article Snippet: Prior to PCR, 5 μl of total DNA from each time point was digested with 20 units of DpnI restriction enzyme (NEB) overnight in a 50-μl reaction volume to eliminate input bacmid DNA.

    Techniques: Real-time Polymerase Chain Reaction, DNA Synthesis, Isolation, Transfection

    ( A ) The click-oligonucleotides used for site directed mutagenesis contained a silent C to A mutation (shown in blue), that introduces a BamHI restriction site not present in the native mCherry gene. The click-linked bases are shown in red. ( B ) Assembly of the click-linked pRSET-mCherry plasmid by site directed mutagenesis, introducing a BamHI watermark. ( C ) Gel electrophoresis (0.8% agarose gel) of SDM products (expected size 3577 bp); Lane 1, 2-log DNA ladder (New England Biolabs); lane 2, pRSET-mCherry SDM with normal primers; lane 3, pRSET-mCherry SDM with normal primers followed by DpnI digestion; lane 4, pRSET-mCherry SDM using click primers; lane 5, pRSET-mCherry SDM using click primers followed by DpnI digestion; lane 6, negative control (pRSET-mCherry SDM using water instead of primers); lane 7, negative control followed by DpnI digestion; lane 8, pRSET-mCherry template plasmid.

    Journal: Nucleic Acids Research

    Article Title: Assessing the biocompatibility of click-linked DNA in Escherichia coli

    doi: 10.1093/nar/gks756

    Figure Lengend Snippet: ( A ) The click-oligonucleotides used for site directed mutagenesis contained a silent C to A mutation (shown in blue), that introduces a BamHI restriction site not present in the native mCherry gene. The click-linked bases are shown in red. ( B ) Assembly of the click-linked pRSET-mCherry plasmid by site directed mutagenesis, introducing a BamHI watermark. ( C ) Gel electrophoresis (0.8% agarose gel) of SDM products (expected size 3577 bp); Lane 1, 2-log DNA ladder (New England Biolabs); lane 2, pRSET-mCherry SDM with normal primers; lane 3, pRSET-mCherry SDM with normal primers followed by DpnI digestion; lane 4, pRSET-mCherry SDM using click primers; lane 5, pRSET-mCherry SDM using click primers followed by DpnI digestion; lane 6, negative control (pRSET-mCherry SDM using water instead of primers); lane 7, negative control followed by DpnI digestion; lane 8, pRSET-mCherry template plasmid.

    Article Snippet: DpnI digestion and purification of SDM products DpnI restriction endonuclease (NEB, Cat. No. R0176L) was directly added to the product of the above amplification reaction, and incubated at room temperature for 6 h. The SDM product was separated from any remaining template by gel purification from 0.8% agarose using the QIAquick Gel Extraction Kit (QIAgen) following the manufacturer’s instructions.

    Techniques: Mutagenesis, Plasmid Preparation, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Negative Control

    Two-step derivation of mutations in E1B and E4. A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

    Journal: PLoS ONE

    Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

    doi: 10.1371/journal.pone.0199563

    Figure Lengend Snippet: Two-step derivation of mutations in E1B and E4. A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

    Article Snippet: Input plasmid DNA templates were digested with 20 U of the restriction enzyme DpnI (New England Biolabs) for 30 min at 37°C.

    Techniques: Mutagenesis, Construct, Amplification, Blocking Assay, Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Modification, FLAG-tag, Infection, Generated

    Overview of the mutagenesis technique. Two PCR reactions are done per mutant, in each of them approximately half of the vector is amplified. Two fragments containing one mutation are combined, followed by DpnI digestion at 37 °C overnight. Reaction clean-up is performed to purify DNA fragments, which are then assembled by Gibson assembly reaction. Bacteria are transformed with the resulting circular plasmid and plated on selective LB agar plates. One clone per mutant is sent for sequencing either on a selective LB agar 96-well plate or as purified DNA. All steps excluding the plating of the bacteria are done in 96-well plates.

    Journal: Scientific Reports

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach

    doi: 10.1038/s41598-017-07010-4

    Figure Lengend Snippet: Overview of the mutagenesis technique. Two PCR reactions are done per mutant, in each of them approximately half of the vector is amplified. Two fragments containing one mutation are combined, followed by DpnI digestion at 37 °C overnight. Reaction clean-up is performed to purify DNA fragments, which are then assembled by Gibson assembly reaction. Bacteria are transformed with the resulting circular plasmid and plated on selective LB agar plates. One clone per mutant is sent for sequencing either on a selective LB agar 96-well plate or as purified DNA. All steps excluding the plating of the bacteria are done in 96-well plates.

    Article Snippet: To reduce background, methylated template DNA was digested by adding 0.5 μL DpnI enzyme (20 units μL−1 from NEB or 10 units μL−1 from Thermo Scientific) to a final PCR mixture.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Transformation Assay, Sequencing, Purification

    Number of colonies obtained for cloning a 1.2 kb insert into a 4.7 kb vector. The plasmid (pETMCSI) [ 21 ] was linearised by PCR using as template (A) undigested and (B) Nde I- Eco RI double-digested plasmid. Digestions with E2 and DpnI were performed at 37 °C for the durations indicated and all experiments were performed in triplicate. (C) Negative control conducted with vector or insert only, following 60 minutes of E2/DpnI digestion. V1: linearized vector. V2: vector double-digested with Nde I and Eco RI prior to linearization by PCR. I: insert.

    Journal: PLoS ONE

    Article Title: Mutant T4 DNA polymerase for easy cloning and mutagenesis

    doi: 10.1371/journal.pone.0211065

    Figure Lengend Snippet: Number of colonies obtained for cloning a 1.2 kb insert into a 4.7 kb vector. The plasmid (pETMCSI) [ 21 ] was linearised by PCR using as template (A) undigested and (B) Nde I- Eco RI double-digested plasmid. Digestions with E2 and DpnI were performed at 37 °C for the durations indicated and all experiments were performed in triplicate. (C) Negative control conducted with vector or insert only, following 60 minutes of E2/DpnI digestion. V1: linearized vector. V2: vector double-digested with Nde I and Eco RI prior to linearization by PCR. I: insert.

    Article Snippet: To 200 ng of purified PCR products in 20 μL in T4 buffer are added 1 μL of 4 μM (0.4 mg/mL) stock solution of E2 enzyme and 1 μL of DpnI restriction enzyme stock (New England Biolabs).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Negative Control

    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation