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    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.
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    New England Biolabs kpni saci dpni
    Inhibition of KSHV ori-Lyt -dependent DNA replication with (+)-rutamarin. BCBL-1 cells were transfected with a KSHV ori-Lyt -containing plasmid (pOri-A) and an RTA expression vector (pCR3.1-ORF50). The transfected cells were treated with increasing concentrations of (+)-rutamarin and incubated for 72 h. Hirt DNAs were extracted and digested with <t>KpnI/SacI</t> or <t>KpnI/SacI/DpnI.</t> DpnI-resistant viral replicated DNA (Rep'd DNA) was detected by Southern blotting with 32 P-labeled pBluescript plasmid.
    Kpni Saci Dpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs endonuclease dpni
    (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: <t>DpnI</t> treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.
    Endonuclease Dpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    ( 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

    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

    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

    Inhibition of KSHV ori-Lyt -dependent DNA replication with (+)-rutamarin. BCBL-1 cells were transfected with a KSHV ori-Lyt -containing plasmid (pOri-A) and an RTA expression vector (pCR3.1-ORF50). The transfected cells were treated with increasing concentrations of (+)-rutamarin and incubated for 72 h. Hirt DNAs were extracted and digested with KpnI/SacI or KpnI/SacI/DpnI. DpnI-resistant viral replicated DNA (Rep'd DNA) was detected by Southern blotting with 32 P-labeled pBluescript plasmid.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Antiviral Activity of (+)-Rutamarin against Kaposi's Sarcoma-Associated Herpesvirus by Inhibition of the Catalytic Activity of Human Topoisomerase II

    doi: 10.1128/AAC.01259-13

    Figure Lengend Snippet: Inhibition of KSHV ori-Lyt -dependent DNA replication with (+)-rutamarin. BCBL-1 cells were transfected with a KSHV ori-Lyt -containing plasmid (pOri-A) and an RTA expression vector (pCR3.1-ORF50). The transfected cells were treated with increasing concentrations of (+)-rutamarin and incubated for 72 h. Hirt DNAs were extracted and digested with KpnI/SacI or KpnI/SacI/DpnI. DpnI-resistant viral replicated DNA (Rep'd DNA) was detected by Southern blotting with 32 P-labeled pBluescript plasmid.

    Article Snippet: The extracted extrachromosomal DNA was treated with RNase A at 25°C for 30 min, followed by proteinase K at 50°C for 30 min. Five micrograms of DNA was digested with KpnI/SacI or KpnI/SacI/DpnI (New England Bio-Labs), separated on a 1% agarose gel, and transferred onto GeneScreen membranes (PerkinElmer, Boston, MA).

    Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Incubation, Southern Blot, Labeling

    (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Polyacrylamide Gel Electrophoresis, Marker