ecori  (New England Biolabs)


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    Structured Review

    New England Biolabs ecori
    Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecori/product/New England Biolabs
    Average 99 stars, based on 353 article reviews
    Price from $9.99 to $1999.99
    ecori - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Diagnostic Assay:

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: The resulting epitope predictions were used collectively to select a TCoV-S1 gene fragment from which to develop a diagnostic ELISA. .. PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA).

    Clone Assay:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: .. This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST. .. The transmembrane domain of Duf was replaced with that of DE-Cadherin and Semaphorin-1a using nested PCR.

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: Paragraph title: Cloning and mutagenesis ... GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively.

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: E. coli recombinant clones were confirmed using colony PCR with the primer pair I0643iF and I0643iR listed in . .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: .. The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector. .. To identify the putative domain of amino acid sequence required for hnRNP A1/SARS_N interaction, different fragments of SARS_N gene were prepared by PCR.

    Article Title: BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.
    Article Snippet: Constructs All new plasmids used are cloned into pcDNA3 vector (Life Technologies). .. The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] , using oligoes described in Supplementary Table 3 .

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: Paragraph title: Selection and cloning of the TCoV-S 54-395 protein gene ... PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA).

    Centrifugation:

    Article Title: Candidate Causal Variants at the 8p12 Breast Cancer Risk Locus Regulate DUSP4.
    Article Snippet: .. Cell nuclei were collected by centrifugation and incubated overnight at 37 ◦C with 1500 U of EcoRI in New England Biolabs restriction buffer with 0.3% sodium dodecyl sulfate and 2% Triton-X 100. .. Restriction enzyme was heat-inactivated at 65 ◦C for 20 min and ligation was performed in 8 mL of ligation buffer (1% Triton X-100, 1.15× NEB ligation buffer, 0.1 mg/mL bovine serum albumin and 1 mM ATP).

    Amplification:

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: Similarly, to generate pcDNA 3.1-puro-mCherry, PCR amplified mCherry was cloned into EcoRI (New England Biolabs) linearized pcDNA 3.1-puro vector. .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively.

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs). ..

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: .. The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector. .. To identify the putative domain of amino acid sequence required for hnRNP A1/SARS_N interaction, different fragments of SARS_N gene were prepared by PCR.

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: The amplified fragment was restricted with MfeI and EcoRI and inserted at the EcoRI site in NR-3ЈNVS. .. Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment.

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: A fragment of the S1 region representing the amino acid positions 54-395 of the entire S glycoprotein was amplified using primers SBETF162 (5 0 -AAAGGATCCTA-GATTTTTATAGTCCAGAT-3 0 ) and SBETR1164 (5 0 -AAA-GAATTCTACACTAACGACATAAACAC-3 0 ). .. PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA).

    DNA Ligation:

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs). ..

    Polymerase Chain Reaction:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: The 2 fragments together served as a template for the next round of PCR using Duf-F and Duf-flag-R. .. This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST.

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively. .. MLLN -GFP, MLLC- GFP, and MLLN -mCherry, constructs were made by PCR cloning MLLN or MLLC into XhoI (New England Biolabs) linearized pcDNA3.1-GFP, or pcDNA3.1-mCherry vectors, high fidelity Phusion polymerase (New England Biolabs) was used for PCR amplification.

    Article Title: Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism *
    Article Snippet: These fragments were obtained by PCR of A. niger ATCC 1015 genomic DNA using primers larA-5-F, larA-5-R, larA-3-F, larA-3-R, pyrG-del-F_n, and pyrG-del-R_n , and the proofreading DNA polymerase Phusion (Finnzymes). .. The larA terminator fragment ( larA -3) digested with EcoRI (NEB) was inserted into the plasmid pRSET-A (Invitrogen), which was digested with EcoRI and PvuII (both NEB).

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: E. coli recombinant clones were confirmed using colony PCR with the primer pair I0643iF and I0643iR listed in . .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector. .. To identify the putative domain of amino acid sequence required for hnRNP A1/SARS_N interaction, different fragments of SARS_N gene were prepared by PCR.

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: Robo502-SIN3ЈUTR, a Robo502 derivative which contains the SIN 3ЈUTR in place of the RUB 3ЈUTR, was constructed by PCR using pTE5ЈZJ/CAT as a template and upstream primer 694 (nts 11,302-11,318 of the SIN genome, including an MfeI site) and downstream primer 695 (EcoRI site followed by a T 20 tract and sequences complementary to the 3Ј-twenty nts of the SIN genome). .. Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment.

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: .. PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA). .. PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit.

    Construct:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: .. This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST. .. The transmembrane domain of Duf was replaced with that of DE-Cadherin and Semaphorin-1a using nested PCR.

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: Complementary DNA (cDNA) construct having full-length MLL was cloned into pBabe-FLAG vector as described [ ]. pcDNA 3.1-puro-GFP was made by inserting polymerase chain reaction (PCR) amplified GFP into KpnI (New England Biolabs) linearized pcDNA 3.1-puro vector. .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively.

    Article Title: Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism *
    Article Snippet: The larA terminator fragment ( larA -3) digested with EcoRI (NEB) was inserted into the plasmid pRSET-A (Invitrogen), which was digested with EcoRI and PvuII (both NEB). .. This intermediary construct was digested with NdeI (NEB), treated with the Klenow polymerase, purified and then digested with SacI (NEB).

    Article Title: BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.
    Article Snippet: Paragraph title: Constructs ... The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] , using oligoes described in Supplementary Table 3 .

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: Paragraph title: Vectors and plasmid constructs ... Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: The resulting epitope predictions were used collectively to select a TCoV-S1 gene fragment from which to develop a diagnostic ELISA. .. PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA).

    Incubation:

    Article Title: Candidate Causal Variants at the 8p12 Breast Cancer Risk Locus Regulate DUSP4.
    Article Snippet: .. Cell nuclei were collected by centrifugation and incubated overnight at 37 ◦C with 1500 U of EcoRI in New England Biolabs restriction buffer with 0.3% sodium dodecyl sulfate and 2% Triton-X 100. .. Restriction enzyme was heat-inactivated at 65 ◦C for 20 min and ligation was performed in 8 mL of ligation buffer (1% Triton X-100, 1.15× NEB ligation buffer, 0.1 mg/mL bovine serum albumin and 1 mM ATP).

    Transformation Assay:

    Article Title: Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism *
    Article Snippet: The deletion cassette, 4930 bp, containing the xyrA flanking regions and the pyrG gene, was released by EcoRI (NEB) digestion and transformed into A. niger ATCC 1015 Δ pyrG . .. The larA terminator fragment ( larA -3) digested with EcoRI (NEB) was inserted into the plasmid pRSET-A (Invitrogen), which was digested with EcoRI and PvuII (both NEB).

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: Ligations were transformed into One Shot chemically competent E. coli TOP10 and screened accordingly ( ). .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA). .. E. coli DH5a competent cells were transformed with the ligation mix and plated onto LB agar plates containing ampicillin (0.1 mg/ml).

    Derivative Assay:

    Article Title: BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.
    Article Snippet: The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] , using oligoes described in Supplementary Table 3 . .. MHC-Iα having C-terminal 2A* and 2A-P* sequence was derived from a previously described MHC-Iα plasmid [39] .

    Inverse PCR:

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively. .. All the PCR cloning was done using In-Fusion HD Cloning Kit (Takara) as per the manufacturer’s instructions. pcDNA-MLLN -mCherry and pcDNAFRT-MLLC -GFP were subjected to inverse PCR using KOD FX high fidelity enzyme (Toyobo KOD-201) to generate MLLN -∆FYRN (∆1989–2102 amino acid) and MLLC-∆ FYRC (∆3671–3755 amino acid) constructs [ ].

    Ligation:

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA). .. Ligation of the digested product with linearized pGEX-4T3 was performed using T4 DNA ligase (Invitrogen, Carlsbad, CA).

    Article Title: Candidate Causal Variants at the 8p12 Breast Cancer Risk Locus Regulate DUSP4.
    Article Snippet: Cell nuclei were collected by centrifugation and incubated overnight at 37 ◦C with 1500 U of EcoRI in New England Biolabs restriction buffer with 0.3% sodium dodecyl sulfate and 2% Triton-X 100. .. Restriction enzyme was heat-inactivated at 65 ◦C for 20 min and ligation was performed in 8 mL of ligation buffer (1% Triton X-100, 1.15× NEB ligation buffer, 0.1 mg/mL bovine serum albumin and 1 mM ATP).

    Generated:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: Two PCR fragments were first generated using the Forward primer for the mutation + Duf-flag-R and the Reverse primer for the mutation + Duf-F. Full length Duf in pCI-neo was used as the template. .. This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST.

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively. .. MLLN -GFP, MLLC- GFP, and MLLN -mCherry, constructs were made by PCR cloning MLLN or MLLC into XhoI (New England Biolabs) linearized pcDNA3.1-GFP, or pcDNA3.1-mCherry vectors, high fidelity Phusion polymerase (New England Biolabs) was used for PCR amplification.

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: .. Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment. .. Vectors and plasmid constructs The standard nonreplicating vector, NR-5355-3Ј, contained the 3Ј end of the Robo cDNA from nt 5355 through the 3Ј EcoRI linearization site following the poly(A) tract in a pGEM plasmid vector under control of the SP6 RNA polymerase promoter.

    Article Title: Candidate Causal Variants at the 8p12 Breast Cancer Risk Locus Regulate DUSP4.
    Article Snippet: C Analysis 3C libraries were generated from MCF-7, T-47D, and Bre-80 cells. .. Cell nuclei were collected by centrifugation and incubated overnight at 37 ◦C with 1500 U of EcoRI in New England Biolabs restriction buffer with 0.3% sodium dodecyl sulfate and 2% Triton-X 100.

    other:

    Article Title: Acute and chronic gregarisation are associated with distinct DNA methylation fingerprints in desert locusts
    Article Snippet: For each sample of genomic DNA, one 500 ng aliquot was digested with EcoRI and MspI by combining 3 μl target DNA, 0.05 μl EcoRI (20,000 units), 0.25 μl MspI (20,000 units), 1 μl 10× NEBuffer 4 and 5.7 μl H2 O; another 500 ng aliquot of genomic DNA was digested with EcoRI and HpaII by combining 3 μl target DNA, 0.5 μl EcoRI (20,000 units), 0.5 μl HpaII (10,000 units), 1 μl 10× NEBuffer 1 and 5.45 μl H2 O at 37 °C for 3 h.

    Y2H Assay:

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: Paragraph title: Recombinant vectors construction for yeast two-hybrid assay ... The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector.

    Sequencing:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST. .. Loner-V5 was generated by cloning the Loner sequence from genomic DNA extracted from pUAST-Loner-Isoform I flies into the EcoRI and XhoI sites of pAC5.1C in frame with the V5epitope tag at the C terminus.

    Article Title: Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism *
    Article Snippet: The resulting plasmid was verified by restriction analysis and sequencing. .. The larA terminator fragment ( larA -3) digested with EcoRI (NEB) was inserted into the plasmid pRSET-A (Invitrogen), which was digested with EcoRI and PvuII (both NEB).

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector. .. To identify the putative domain of amino acid sequence required for hnRNP A1/SARS_N interaction, different fragments of SARS_N gene were prepared by PCR.

    Article Title: BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.
    Article Snippet: The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] , using oligoes described in Supplementary Table 3 . .. MHC-Iα having C-terminal 2A* and 2A-P* sequence was derived from a previously described MHC-Iα plasmid [39] .

    Recombinant:

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: E. coli recombinant clones were confirmed using colony PCR with the primer pair I0643iF and I0643iR listed in . .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: Paragraph title: Recombinant vectors construction for yeast two-hybrid assay ... The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector.

    Mutagenesis:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: Two PCR fragments were first generated using the Forward primer for the mutation + Duf-flag-R and the Reverse primer for the mutation + Duf-F. Full length Duf in pCI-neo was used as the template. .. This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST.

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: Paragraph title: Cloning and mutagenesis ... GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively.

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: Paragraph title: Construction of a B. thailandensis DW503 recA mutant. ... Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.
    Article Snippet: The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] , using oligoes described in Supplementary Table 3 . .. When indicated, SV5-tag (GKPIPNPLLGLD), roTag (SISSSIFKNEG) [61] , HA-tag (YPYDVPDYA) and N-glycosylation site (NGT) were engineered at the indicated positions by site-directed mutagenesis (QuikChange Site Directed Mutagenesis Kit; Stratagene).

    Isolation:

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: .. Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment. .. Vectors and plasmid constructs The standard nonreplicating vector, NR-5355-3Ј, contained the 3Ј end of the Robo cDNA from nt 5355 through the 3Ј EcoRI linearization site following the poly(A) tract in a pGEM plasmid vector under control of the SP6 RNA polymerase promoter.

    Subcloning:

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively. .. MLLN -GFP, MLLC- GFP, and MLLN -mCherry, constructs were made by PCR cloning MLLN or MLLC into XhoI (New England Biolabs) linearized pcDNA3.1-GFP, or pcDNA3.1-mCherry vectors, high fidelity Phusion polymerase (New England Biolabs) was used for PCR amplification.

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs). ..

    Purification:

    Article Title: Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism *
    Article Snippet: The larA terminator fragment ( larA -3) digested with EcoRI (NEB) was inserted into the plasmid pRSET-A (Invitrogen), which was digested with EcoRI and PvuII (both NEB). .. This intermediary construct was digested with NdeI (NEB), treated with the Klenow polymerase, purified and then digested with SacI (NEB).

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: PCRs were cleaned by using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA). .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: .. PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA). .. PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: Selection and cloning of the TCoV-S 54-395 protein gene RT-PCR was performed as previously described (Gomaa et al., 2008a) . .. PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA).

    Nested PCR:

    Article Title: The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner
    Article Snippet: This single fragment was cloned into the EcoRI and NotI sites of pCI-neo following which the full length construct was excised using NheI, blunted with Calf Intestinal Phosphatase (CIP)(New England Biolabs, NEB) and NotI and cloned into the EcoRV and NotI sites of pAc5.1C or the EcoRI (blunted with CIP) and NotI sites of pUAST. .. The transmembrane domain of Duf was replaced with that of DE-Cadherin and Semaphorin-1a using nested PCR.

    Plasmid Preparation:

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively. .. MLLN -GFP, MLLC- GFP, and MLLN -mCherry, constructs were made by PCR cloning MLLN or MLLC into XhoI (New England Biolabs) linearized pcDNA3.1-GFP, or pcDNA3.1-mCherry vectors, high fidelity Phusion polymerase (New England Biolabs) was used for PCR amplification.

    Article Title: Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism *
    Article Snippet: .. The larA terminator fragment ( larA -3) digested with EcoRI (NEB) was inserted into the plasmid pRSET-A (Invitrogen), which was digested with EcoRI and PvuII (both NEB). .. This intermediary construct was digested with NdeI (NEB), treated with the Klenow polymerase, purified and then digested with SacI (NEB).

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs). ..

    Article Title: The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
    Article Snippet: .. The amplified products were digested with EcoRI and BamHI (NEB) then cloned into pGBKT7 vector. .. To identify the putative domain of amino acid sequence required for hnRNP A1/SARS_N interaction, different fragments of SARS_N gene were prepared by PCR.

    Article Title: BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.
    Article Snippet: Constructs All new plasmids used are cloned into pcDNA3 vector (Life Technologies). .. The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] , using oligoes described in Supplementary Table 3 .

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: .. Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment. .. Vectors and plasmid constructs The standard nonreplicating vector, NR-5355-3Ј, contained the 3Ј end of the Robo cDNA from nt 5355 through the 3Ј EcoRI linearization site following the poly(A) tract in a pGEM plasmid vector under control of the SP6 RNA polymerase promoter.

    Real-time Polymerase Chain Reaction:

    Article Title: Candidate Causal Variants at the 8p12 Breast Cancer Risk Locus Regulate DUSP4.
    Article Snippet: Cell nuclei were collected by centrifugation and incubated overnight at 37 ◦C with 1500 U of EcoRI in New England Biolabs restriction buffer with 0.3% sodium dodecyl sulfate and 2% Triton-X 100. .. 3C interactions were quantified by qPCR with primers designed with the EcoRI restriction fragments spanning the 8p12 risk locus (Supplementary Table S2). qPCR was performed using a RotorGene 6000 with a reaction mix containing MyTaq HS DNA polymerase and the addition of 5 mM Syto9.

    Selection:

    Article Title: Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.
    Article Snippet: Paragraph title: Selection and cloning of the TCoV-S 54-395 protein gene ... PCR products were purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA).

    Agarose Gel Electrophoresis:

    Article Title: Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing
    Article Snippet: PCRs were performed using a FailSafe kit with buffer “J” (Epicentre Technologies, Madison, WI) and resolved on a 0.8% agarose gel. .. Gene disruption cassettes were made by digesting pCR2.1-TOPO containing the B. thailandensis recA internal gene amplicon (419 bp) with EcoRI (New England BioLabs, Ipswich, MA), followed by subcloning (Fast-Link DNA ligation kit; Epicentre Technologies) into the suicide vector pTSV3 digested with EcoRI and treated with shrimp alkaline phosphatase (New England BioLabs).

    Article Title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
    Article Snippet: .. Finally, Robo302 5Ј-7318, which terminates at nt 7318, was generated by digesting Robo302 with AscI and EcoRI [the linearization site following the poly (A) tract], treating the digested fragments with Klenow (New England Biolabs) to produce blunt ends, isolation of the larger DNA fragment containing the vector, and cDNA sequences from the 5Ј end through the AscI site by agarose gel electrophoresis, and self-ligating this fragment. .. Vectors and plasmid constructs The standard nonreplicating vector, NR-5355-3Ј, contained the 3Ј end of the Robo cDNA from nt 5355 through the 3Ј EcoRI linearization site following the poly(A) tract in a pGEM plasmid vector under control of the SP6 RNA polymerase promoter.

    Marker:

    Article Title: Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein
    Article Snippet: .. GFP-tagged pcDNA5/FRT and pcDNA5/FRT TO vector, having hygromycin as selectable marker, was generated by PCR sub-cloning of GFP into EcoRI and EcoRV (New England Biolabs) site of pcDNA5/FRT, Xho-1 site of pcDNA5/FRT TO vector respectively. .. MLLN -GFP, MLLC- GFP, and MLLN -mCherry, constructs were made by PCR cloning MLLN or MLLC into XhoI (New England Biolabs) linearized pcDNA3.1-GFP, or pcDNA3.1-mCherry vectors, high fidelity Phusion polymerase (New England Biolabs) was used for PCR amplification.

    Lysis:

    Article Title: Candidate Causal Variants at the 8p12 Breast Cancer Risk Locus Regulate DUSP4.
    Article Snippet: After washing with PBS, cells were incubated in ice-cold cell lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.2% IGEPAL and cOmplete protease inhibitors (Roche)) for 30 min on ice, followed by 10 strokes of a Dounce homogenizer. .. Cell nuclei were collected by centrifugation and incubated overnight at 37 ◦C with 1500 U of EcoRI in New England Biolabs restriction buffer with 0.3% sodium dodecyl sulfate and 2% Triton-X 100.

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    New England Biolabs restriction enzymes ecori
    The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with <t>AvaI</t> and <t>EcoRI.</t> The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown
    Restriction Enzymes Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Journal: Journal of industrial microbiology & biotechnology

    Article Title: Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

    doi: 10.1007/s10295-018-2049-x

    Figure Lengend Snippet: The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Article Snippet: The colonies were picked into 10 mL LB with 50 μg/mL apramycin, and the plasmid DNA was extracted using a Miniprep kit (Qiagen, Valencia, CA, USA) and screened with restriction enzymes EcoRI and AvaI for pJGW92 and NcoI and AvaI for pJGW93 (NEB).

    Techniques: Plasmid Preparation, Derivative Assay, Purification, Isolation, Transformation Assay, Polymerase Chain Reaction, Molecular Weight

    Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Journal: PLoS ONE

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    doi: 10.1371/journal.pone.0106713

    Figure Lengend Snippet: Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Article Snippet: We then double-digest ∼1.0 µg of DNA with high fidelity versions of the SbfI and EcoRI restriction enzymes (New England Biolabs); when less DNA is available, we have had good success starting with as little as 0.17 µg of genomic DNA.

    Techniques: Sequencing

    Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Journal: PLoS ONE

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    doi: 10.1371/journal.pone.0106713

    Figure Lengend Snippet: Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Article Snippet: We then double-digest ∼1.0 µg of DNA with high fidelity versions of the SbfI and EcoRI restriction enzymes (New England Biolabs); when less DNA is available, we have had good success starting with as little as 0.17 µg of genomic DNA.

    Techniques: Sequencing, Isolation, Agarose Gel Electrophoresis

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1.

    Techniques: Southern Blot, Incubation, Generated

    RE digestion of TE-PCR product with EcoRI, Pst I, Eco91l and HindIII . [ Lane M 100 bp DNA ladder; Lane 1 TE-PCR product with out RE; Lane 2 TE-PCR product with EcoRI ; Lane 3 TE-PCR product with Pst I ; Lane 5 TE-PCR product with out RE; Lane 6

    Journal: Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology

    Article Title: Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates

    doi: 10.1007/s12639-014-0582-8

    Figure Lengend Snippet: RE digestion of TE-PCR product with EcoRI, Pst I, Eco91l and HindIII . [ Lane M 100 bp DNA ladder; Lane 1 TE-PCR product with out RE; Lane 2 TE-PCR product with EcoRI ; Lane 3 TE-PCR product with Pst I ; Lane 5 TE-PCR product with out RE; Lane 6

    Article Snippet: EcoRI, Eco91l, HindIII and PstI, for complete digestion with the recommended RE buffers in separate PCR tubes in the following reaction volume: 7 µl nuclease-free water, 10 µl DNA, 2 µl 10× RE buffer, 1 µl (10 U) EcoRI (New England Biolabs)/1 µl (10 U) Eco91l (Fermentas) / 1 µl (10 U) HindIII (Fermentas) / 1 µl (10 U) PstI (Fermentas).

    Techniques: Polymerase Chain Reaction