t5 exonuclease  (New England Biolabs)


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    Name:
    T5 Exonuclease
    Description:

    Catalog Number:
    M0363L
    Price:
    None
    Score:
    85
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    Structured Review

    New England Biolabs t5 exonuclease
    In vitro HBV infection of rhesus macaque PH.  a  Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP.  b  Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488.  c  Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments.  d  HBV DNA qPCR on the same supernatants shown in  c . Each condition represents a single-biological sample ( N  = 1).  e  Total intracellular DNA from 1 × 10 6  rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates.  f  Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments.  g  Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4  or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)

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    Images

    1) Product Images from "Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques"

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01953-y

    In vitro HBV infection of rhesus macaque PH.  a  Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP.  b  Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488.  c  Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments.  d  HBV DNA qPCR on the same supernatants shown in  c . Each condition represents a single-biological sample ( N  = 1).  e  Total intracellular DNA from 1 × 10 6  rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates.  f  Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments.  g  Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4  or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)
    Figure Legend Snippet: In vitro HBV infection of rhesus macaque PH. a Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP. b Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488. c Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments. d HBV DNA qPCR on the same supernatants shown in c . Each condition represents a single-biological sample ( N  = 1). e Total intracellular DNA from 1 × 10 6 rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates. f Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments. g Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4 or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)

    Techniques Used: In Vitro, Infection, Labeling, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Southern Blot, Purification, Marker

    2) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    3) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    4) Product Images from "Patch cloning method for multiple site-directed and saturation mutagenesis"

    Article Title: Patch cloning method for multiple site-directed and saturation mutagenesis

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-91

    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
    Figure Legend Snippet: Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation

    5) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    Journal: Genes and Environment

    doi: 10.1186/s41021-018-0112-5

    Preparation of DNA template (A:C mismatch substrate) with A:C mismatch at a defined position.  a  Upper strand sequence containing A base and lower strand substrate containing original C base in pBS2/A:C are shown diagrammatically.  b  Experimental procedure for purification of pBS2/A:C.  c  Aliquots from various steps of the purification were analyzed on 0.8% agarose gel, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4,  Eco NI-treatment; lane 5, T5 exonuclease-treatment. Lower panel shows final purified DNA products (%). Open circular DNA (OC), linear DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrow.  d  Upper strand sequence containing A base in pBS2/A:C was sequenced. An A:C mismatch site is indicated by the arrow.  e  Lower strand sequence containing base A in pBS2/A:C was sequenced. The A:C mismatch site is indicated by the arrow
    Figure Legend Snippet: Preparation of DNA template (A:C mismatch substrate) with A:C mismatch at a defined position. a Upper strand sequence containing A base and lower strand substrate containing original C base in pBS2/A:C are shown diagrammatically. b Experimental procedure for purification of pBS2/A:C. c Aliquots from various steps of the purification were analyzed on 0.8% agarose gel, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment. Lower panel shows final purified DNA products (%). Open circular DNA (OC), linear DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrow. d Upper strand sequence containing A base in pBS2/A:C was sequenced. An A:C mismatch site is indicated by the arrow. e Lower strand sequence containing base A in pBS2/A:C was sequenced. The A:C mismatch site is indicated by the arrow

    Techniques Used: Sequencing, Purification, Agarose Gel Electrophoresis, Staining, Countercurrent Chromatography

    a Experimental design. The plasmid pBS2-SDL was digested with a nicking endonuclease. An oligonucleotide containing a DNA lesion was hybridized with gap plasmid and ligated using T4 DNA ligase. Original plasmids in the sample are digested with restriction enzymes, except for DNA lesion bearing plasmids. T5 exonuclease cuts only the linear DNA plasmids digested by Eco NI, and does not work on sealed DNA plasmids containing a DNA lesion. b Covalently closed circular duplex DNA containing a single lesion. Sixty four-basepair oligonucleotides containing a single DNA lesion site within the Eco NI restriction enzyme site, two nicking endonuclease sites and the plasmid pBS2-SDL (2917 bp) are shown diagrammatically
    Figure Legend Snippet: a Experimental design. The plasmid pBS2-SDL was digested with a nicking endonuclease. An oligonucleotide containing a DNA lesion was hybridized with gap plasmid and ligated using T4 DNA ligase. Original plasmids in the sample are digested with restriction enzymes, except for DNA lesion bearing plasmids. T5 exonuclease cuts only the linear DNA plasmids digested by Eco NI, and does not work on sealed DNA plasmids containing a DNA lesion. b Covalently closed circular duplex DNA containing a single lesion. Sixty four-basepair oligonucleotides containing a single DNA lesion site within the Eco NI restriction enzyme site, two nicking endonuclease sites and the plasmid pBS2-SDL (2917 bp) are shown diagrammatically

    Techniques Used: Plasmid Preparation

    One-pot synthesis of DNA repair substrate.  a  Experimental procedure for purification of pBS2/A:C omitting a column purification step.  b  Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4,  Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.
    Figure Legend Snippet: One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Countercurrent Chromatography

    6) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    7) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    8) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    9) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    10) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    11) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    12) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Lesion-containing construct quality controls. (A) Schematic of the Fpg nicking assay. Fpg cleaves damages, such as 8-oxoG and 5-OHU, leaving a single-strand break, converting the construct from covalently closed (cc) to nicked form. (B) Lesion structures. (C) Representative images of Fpg and Nth nicked T5 exonuclease-treated lesion-containing and lesion-free control constructs. Fpg cleaves 8-oxoG, 5-OHU, and DHU, nicking the lesion-containing constructs almost entirely, but not the lesion-free controls, and Nth cleaves DHU.
    Figure Legend Snippet: Lesion-containing construct quality controls. (A) Schematic of the Fpg nicking assay. Fpg cleaves damages, such as 8-oxoG and 5-OHU, leaving a single-strand break, converting the construct from covalently closed (cc) to nicked form. (B) Lesion structures. (C) Representative images of Fpg and Nth nicked T5 exonuclease-treated lesion-containing and lesion-free control constructs. Fpg cleaves 8-oxoG, 5-OHU, and DHU, nicking the lesion-containing constructs almost entirely, but not the lesion-free controls, and Nth cleaves DHU.

    Techniques Used: Construct

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT  T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG  constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT  construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    Optimization for DNA integrity and mammalian transfection. (A) Schematic representing T5 exonuclease digestion of nicked, linear, and ssDNA. (B) Representative gel electrophoresis of a construct with and without T5 exonuclease treatment prior to purification and after purification. (C) Construct yields after T5 exonuclease treatment after initial purification (after) or directly in the second strand synthesis reaction (before), relative to non-T5 exonuclease treated construct (none). Error bars represent the standard deviation. (D) Live cell images of Ogg1 -/-  MEFs nucleofected with EGFP construct treated or not treated with T5 exonuclease or EGFP bacterial plasmid maxiprep, and stained with Hoechst 33342 dye. T5 exonuclease digestion of nicked and linear construct does not improve transfection efficiencies.
    Figure Legend Snippet: Optimization for DNA integrity and mammalian transfection. (A) Schematic representing T5 exonuclease digestion of nicked, linear, and ssDNA. (B) Representative gel electrophoresis of a construct with and without T5 exonuclease treatment prior to purification and after purification. (C) Construct yields after T5 exonuclease treatment after initial purification (after) or directly in the second strand synthesis reaction (before), relative to non-T5 exonuclease treated construct (none). Error bars represent the standard deviation. (D) Live cell images of Ogg1 -/- MEFs nucleofected with EGFP construct treated or not treated with T5 exonuclease or EGFP bacterial plasmid maxiprep, and stained with Hoechst 33342 dye. T5 exonuclease digestion of nicked and linear construct does not improve transfection efficiencies.

    Techniques Used: Transfection, Nucleic Acid Electrophoresis, Construct, Purification, Standard Deviation, Plasmid Preparation, Staining

    13) Product Images from "QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides"

    Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175146

    Circularization of linear plasmid library and removal of starting matrix. (a) Time course of the circularization reaction: a mix of four enzymes (T5 exonuclease, DNA polymerase, DNA ligase and DpnI) was added to the amplified linear plasmids and incubated for one hour at 50 C. The amount of T5 exonuclease was reduced 4-fold compared to Gibson’s protocol. Circularization products (left side) are also analyzed by restriction with AvaI (right side). The band at 3.6 kb (black arrow) is only present when the plasmids are sealed. (b) DpnI is cleaving the starting matrix at 50 C while the synthetic PCR product is resistant to its action.
    Figure Legend Snippet: Circularization of linear plasmid library and removal of starting matrix. (a) Time course of the circularization reaction: a mix of four enzymes (T5 exonuclease, DNA polymerase, DNA ligase and DpnI) was added to the amplified linear plasmids and incubated for one hour at 50 C. The amount of T5 exonuclease was reduced 4-fold compared to Gibson’s protocol. Circularization products (left side) are also analyzed by restriction with AvaI (right side). The band at 3.6 kb (black arrow) is only present when the plasmids are sealed. (b) DpnI is cleaving the starting matrix at 50 C while the synthetic PCR product is resistant to its action.

    Techniques Used: Plasmid Preparation, Amplification, Incubation, Polymerase Chain Reaction

    14) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    15) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    16) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    17) Product Images from "Unique nucleotide sequence (UNS)-guided assembly of repetitive DNA parts for synthetic biology applications"

    Article Title: Unique nucleotide sequence (UNS)-guided assembly of repetitive DNA parts for synthetic biology applications

    Journal:

    doi: 10.1038/nprot.2014.145

    Sequence considerations when using standard part vectors When using currently available part vectors , restriction sites lying just outside of the UNSs are digested to generate linear parts. Shown is an example in which pFLU1U2 containing “Part A” is digested with AscI and MauBI. This digestion leaves behind terminal nucleotides from the restriction sites (orange). The 5′ nucleotides at the termini are digested by T5 exonuclease during isothermal assembly, but those at the 3′ ends are left behind. When Part A anneals to another part, “Part B” (bottom), the terminal nucleotides in Part A anneal with nucleotides internal to the UNSs in Part B and vice versa . Caution must be taken to ensure these terminal nucleotides do not create mismatches that might interfere with assembly.
    Figure Legend Snippet: Sequence considerations when using standard part vectors When using currently available part vectors , restriction sites lying just outside of the UNSs are digested to generate linear parts. Shown is an example in which pFLU1U2 containing “Part A” is digested with AscI and MauBI. This digestion leaves behind terminal nucleotides from the restriction sites (orange). The 5′ nucleotides at the termini are digested by T5 exonuclease during isothermal assembly, but those at the 3′ ends are left behind. When Part A anneals to another part, “Part B” (bottom), the terminal nucleotides in Part A anneal with nucleotides internal to the UNSs in Part B and vice versa . Caution must be taken to ensure these terminal nucleotides do not create mismatches that might interfere with assembly.

    Techniques Used: Sequencing

    18) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    19) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    20) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    Related Articles

    Clone Assay:

    Article Title: Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases
    Article Snippet: Paragraph title: Cloning ... T5 exonuclease, Q5 High-Fidelity DNA Polymerase and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA).

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: Overlapping sequences (20 nucleotides [nt]) were inserted into the primers to the adjacent sequences of interest for cloning using the one-step isothermal assembly method ( ). .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl.

    Centrifugation:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: After centrifugation, supernatants were collected and further treated with DNase I and RNase A. NCs were then digested with proteinase K and sodium dodecyl sulfate (SDS). .. The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions.

    Amplification:

    Article Title: Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases
    Article Snippet: T5 exonuclease, Q5 High-Fidelity DNA Polymerase and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA). .. PCR reactions were run in a Bio-Rad S1000 Thermal Cycler.

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: The amyE integration vector pPG40 ( ) containing the β-galactosidase gene ( lacZ ) was amplified with the LB139-LB140 and LB143-LB144 primer pairs for cloning PrecA and PyneA, respectively. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl.

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert. .. The 150 bp DNA was produced with a 50-μL PCR using the same reaction solution conditions as above and the following temperature regime (time in mm:ss): [98 °C 02:00 | 5 × (98 °C 00:10 | 50 °C 00:10 | 68 °C 04:00) | 15 × (98 °C 00:10 | 58 °C 00:10 | 68 °C 04:00)].

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions. .. The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions.

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: The upstream homologous arm was PCR amplified using primers Fup and Rup, and the downstream homologous arm amplified by Fhfq-1, Fhfq-2, and Rhfq. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: In short, the library was amplified using gene specific Gibson adaptor primers ( ) bearing an 18 base pair extension that overlaps with the RTS upstream and RTS downstream fragments. .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB).

    Synthesized:

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert.

    Neutralization:

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The adaptor-ligated DNA fragments were incubated in 0.3 M NaOH for 2 hr at 55 °C to expose 2′,3′-cyclic phosphate and 2′-phosphate termini of DNA at rNMP sites, followed by neutralization and purification. .. The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact.

    Construct:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: Higher yields can be obtained if the T5 exonuclease treatment is performed directly in the second strand synthesis reaction ( ). .. We find that treatment with T5 exonuclease does not improve transfection efficiencies, and EGFP constructs treated or not treated with the enzyme result in similar efficiencies ( ). .. We also found that bacterial EGFP plasmid purified using the same method and of identical purity results in higher efficiencies than both types of constructs, likely due to differences in the plasmid coiling.

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: The resulting plasmid was verified by sequencing and transformed into B. subtilis competent cells, resulting in strain UG10. recA and yneA promoter reporters were constructed by amplifying the promoter regions with the LB141-LB142 and LB145-LB146 primer pairs, respectively, and using the genomic DNA of strain 168 as the template. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl.

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: pINFs ( ) were constructed through Golden Gate assembly of pUCX2 and insert dsDNA containing a d NaM -d TPT3 pair as described previously with the following modifications. .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    SYBR Green Assay:

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs. .. This protocol was shown to specifically allow the detection of cccDNA and to avoid contamination by rcDNA (Qu et al ., manuscript in preparation). β-globin expression levels from undigested samples were used for normalization (β-globin-F; β-globin-R).

    Modification:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: The cccDNA purification was performed using a modified Hirt extraction procedure [ ]. .. The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions.

    Incubation:

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: For pINF assembly, pUCX2 (1 μg) and insert DNA were combined at a 1:4 molar ratio in a 80 μL reaction with ATP (1 mM), T4 DNA ligase (6.65 U/μL, New England Biolabs), BsaI-HF (0.66 U/μL, New England Biolabs), and CutSmart Buffer (1×, New England Biolabs) and subjected to the following temperature regime: [37 °C 20 min | 40 × (37 °C 5 min | 16 °C 10 min | 22 °C 5 min) | 37 °C 20 min | 50 °C 15 min | 70 °C 30 min]. .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert. .. This reaction was purified using a DNA Clean & Concentrator-5 according to manufacturer recommendations except that reactions were mixed with 3 volumes of 1:1 DNA Wash:DNA Binding Buffer before binding to the silica column.

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The resulting single-stranded (ss) fragments were incubated in 50 mM Tris-HCl, pH 7.5, 40 mM NaCl, 5 mM MgCl2 , 1 mM DTT, 30 µM ATP (Sigma-Aldrich), and 1 µM AtRNL for 1 hr at 30 °C, followed by purification. .. The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact.

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: Briefly, cells were trypsinized and re-suspended in provided T1 buffer and incubated with proteinase K and B3 buffer at 70 °C for 1 hour and following steps were stick to the protocol. .. For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs.

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: In short, the library was amplified using gene specific Gibson adaptor primers ( ) bearing an 18 base pair extension that overlaps with the RTS upstream and RTS downstream fragments. .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Reactions were incubated for 1 hr at 50°C and the extent of assembly (typically greater than 90%) confirmed by gel electrophoresis.

    Introduce:

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Primers Fsp, Rsp-1, and Rsp-2 were used to introduce a terminator at the end of the spectinomycin resistance gene and primers Fhfq-1 and Fhfq-2 were used to introduce the 3xFLAG. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Expressing:

    Article Title: Cellular reagents for diagnostics and synthetic biology
    Article Snippet: Lyophilized cellular reagents were prepared as follows for use in Gibson assemblies. .. 2x108 Top10 lyophilized cellular reagents expressing Taq DNA polymerase, T5 exonuclease, or Taq DNA ligase were rehydrated using 30 μl of water. .. The rehydrated T5 exonuclease cellular reagent was diluted 1:100 in water followed by addition of 1.5 μl aliquot per Gibson reaction.

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl. .. The parA single mutant (strain HM161) was constructed elsewhere ( ) and was transformed into our laboratory wild-type strain to ensure an isogenic background (strain KS383).

    Article Title: Cellular reagents for diagnostics and synthetic biology
    Article Snippet: Assemblies using pure enzymes contained 0.08 units of T5 exonuclease (NEB), 0.5 units of Phusion DNA polymerase (NEB) and 80 units of Taq DNA ligase (NEB). .. Assemblies using pure enzymes contained 0.08 units of T5 exonuclease (NEB), 0.5 units of Phusion DNA polymerase (NEB) and 80 units of Taq DNA ligase (NEB).

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs. .. Products were subjected for qPCR with cccDNA specific primers spanning the gap region).

    Knock-In:

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert. .. This reaction was purified using a DNA Clean & Concentrator-5 according to manufacturer recommendations except that reactions were mixed with 3 volumes of 1:1 DNA Wash:DNA Binding Buffer before binding to the silica column.

    Transformation Assay:

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: The resulting plasmid was verified by sequencing and transformed into B. subtilis competent cells, resulting in strain UG10. recA and yneA promoter reporters were constructed by amplifying the promoter regions with the LB141-LB142 and LB145-LB146 primer pairs, respectively, and using the genomic DNA of strain 168 as the template. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl.

    Article Title: Cellular reagents for diagnostics and synthetic biology
    Article Snippet: Paragraph title: Gibson assembly and transformation of chemically competent bacteria ... Assemblies using pure enzymes contained 0.08 units of T5 exonuclease (NEB), 0.5 units of Phusion DNA polymerase (NEB) and 80 units of Taq DNA ligase (NEB).

    Gel Purification:

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Transfection:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: Higher yields can be obtained if the T5 exonuclease treatment is performed directly in the second strand synthesis reaction ( ). .. We find that treatment with T5 exonuclease does not improve transfection efficiencies, and EGFP constructs treated or not treated with the enzyme result in similar efficiencies ( ). .. We also found that bacterial EGFP plasmid purified using the same method and of identical purity results in higher efficiencies than both types of constructs, likely due to differences in the plasmid coiling.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    Southern Blot:

    Article Title: The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection
    Article Snippet: Paragraph title: Southern blotting ... A total of 1 μg of the extracted DNA was digested with 10 units of T5 exonuclease (New England Biolabs, Ipswich, MA) for 2 hours at 37°C.

    Ligation:

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB).

    Infection:

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: For analysis of intracellular total HBV DNA and cccDNA from infected HepG2-NTCP cells, DNA from total cell lysates was prepared using the NucleoSpin Tissue Kit (Macherey-Nagel 740952.250) according to manufacturer’s protocol with minor modifications. .. For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs.

    other:

    Article Title: Cellular reagents for diagnostics and synthetic biology
    Article Snippet: To most simply carry out Gibson assembly with cellular reagents we merely lyophilized three cell lines that expressed Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease, respectively.

    Article Title: Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning
    Article Snippet: All the enzymes including type II restriction enzymes (EcoRI, BamHI, SalI and Bau36I), type IIS restriction enzymes (BsaI, BbsI and BsmBI), T4 DNA ligase, Phusion enzyme, and T5 exonuclease were purchased from New England BioLabs.

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: After dilution, the resulting products were treated with T5 exonuclease for 2 hr at 37 °C.

    Sequencing:

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: Overlapping sequences (20 nucleotides [nt]) were inserted into the primers to the adjacent sequences of interest for cloning using the one-step isothermal assembly method ( ). .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl. .. The reaction mixtures were incubated at 50°C for 1 h. The resulting plasmids pLB74 and pLB75 were verified by restriction enzyme digestion and sequencing and transformed into B. subtilis competent cells, resulting in strains LB641 and LB642, respectively.

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact. .. The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact.

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL). .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    DNA Extraction:

    Article Title: Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes
    Article Snippet: Quantitative PCR for HBV were performed using HBV specific primers and normalized to PRNP housekeeping gene as previously described . .. After total DNA isolation and T5 exonuclease digestion (NEB, Fance) digestion for 1 h, cccDNA was quantified as previously described . .. Alternatively, cccDNA was analyzed by Southern blot after Hirt extraction as described previously .

    Nucleic Acid Electrophoresis:

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Real-time Polymerase Chain Reaction:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: Paragraph title: qPCR and RT-qPCR ... The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions.

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: Paragraph title: Preparation of viral DNA and quantitative PCR analysis ... For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs.

    Mutagenesis:

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl. .. The reaction mixtures were incubated at 50°C for 1 h. The resulting plasmids pLB74 and pLB75 were verified by restriction enzyme digestion and sequencing and transformed into B. subtilis competent cells, resulting in strains LB641 and LB642, respectively.

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Paragraph title: Construction of hfq mutant strains ... Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB).

    Isolation:

    Article Title: The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection
    Article Snippet: DNA was isolated from the nuclei of HBV-infected PHH by Hirt extraction as previously described [ ]. .. A total of 1 μg of the extracted DNA was digested with 10 units of T5 exonuclease (New England Biolabs, Ipswich, MA) for 2 hours at 37°C.

    Polymerase Chain Reaction:

    Article Title: Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases
    Article Snippet: T5 exonuclease, Q5 High-Fidelity DNA Polymerase and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA). .. T5 exonuclease, Q5 High-Fidelity DNA Polymerase and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA).

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: The PCR product and the amyE integration vector pSG1729 ( ) were digested with XhoI and EcoRI restriction enzymes and ligated. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl.

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert.

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact. .. Treatment with the 1 µM Tpt1 in 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.1 mM DTT, 0.4% Triton X-100, and 10 mM NAD+ (Sigma-Aldrich) for 1 hr at 30 °C was used to remove the 2′-phosphate remaining at the ligation junction.

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: The upstream homologous arm was PCR amplified using primers Fup and Rup, and the downstream homologous arm amplified by Fhfq-1, Fhfq-2, and Rhfq. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB).

    Purification:

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: The resulting UBP containing dsDNA was purified using a DNA Clean & Concentrator-5 (Zymo Research) according to manufacturer recommendations. .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert.

    Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides
    Article Snippet: Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs. .. Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs.

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: The cccDNA purification was performed using a modified Hirt extraction procedure [ ]. .. The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions. .. Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The resulting single-stranded (ss) fragments were incubated in 50 mM Tris-HCl, pH 7.5, 40 mM NaCl, 5 mM MgCl2 , 1 mM DTT, 30 µM ATP (Sigma-Aldrich), and 1 µM AtRNL for 1 hr at 30 °C, followed by purification. .. The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL).

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: In short, the library was amplified using gene specific Gibson adaptor primers ( ) bearing an 18 base pair extension that overlaps with the RTS upstream and RTS downstream fragments. .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Reactions were incubated for 1 hr at 50°C and the extent of assembly (typically greater than 90%) confirmed by gel electrophoresis.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
    Article Snippet: Total HBV DNA and RNA were quantified using the TaqMan Fast Advanced Master Mix (Applied Biosystems) and AgPath-ID One-Step RT-PCR kit (Life Technologies), respectively. .. DNA was eluted from NucleoSpin Tissue Columns with 100 μl pre-warmed (70 °C) buffer and subjected to T5 exonuclease treatment (New England Biolabs) at 37 °C for 30 min.

    Quantitative RT-PCR:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: Paragraph title: qPCR and RT-qPCR ... The Hirt-extracted DNA was purified and treated with T5 exonuclease (New England Biolabs) to digest linear and open circular DNA according to the manufacturer’s instructions.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact. .. The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact.

    Plasmid Preparation:

    Article Title: Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases
    Article Snippet: 10G Hi-Control and BL21(DE3) Hi-Control E.coli were obtained from an Expresso T7 cloning kit (Lucigen). pET24a vector was purchased from EMD Millipore. .. T5 exonuclease, Q5 High-Fidelity DNA Polymerase and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA).

    Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis
    Article Snippet: The amyE integration vector pPG40 ( ) containing the β-galactosidase gene ( lacZ ) was amplified with the LB139-LB140 and LB143-LB144 primer pairs for cloning PrecA and PyneA, respectively. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with T5 exonuclease (New England BioLabs [NEB]), Phusion high-fidelity DNA polymerase (NEB), and Taq DNA ligase (NEB) in a total volume of 10 μl.

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert.

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: Absolute copy numbers were calculated referring to a standard based on the linearized HBV coding plasmid pJO19. .. For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts
    Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U T5 exonuclease (NEB, USA), 0.5 μL Buffer 4 (NEB, USA), and nuclease-free water (up to 5 μL). .. The reaction mix was kept on ice for 5 min and then transformed into E. coli competent cells by chemical transformation.

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
    Article Snippet: Quantification was assessed relative to an absolute standard curve using the plasmid pCEP4 with the targeted insert as template. .. DNA was eluted from NucleoSpin Tissue Columns with 100 μl pre-warmed (70 °C) buffer and subjected to T5 exonuclease treatment (New England Biolabs) at 37 °C for 30 min.

    Electrophoresis:

    Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides
    Article Snippet: Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs. .. Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs.

    Agarose Gel Electrophoresis:

    Article Title: The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection
    Article Snippet: A total of 1 μg of the extracted DNA was digested with 10 units of T5 exonuclease (New England Biolabs, Ipswich, MA) for 2 hours at 37°C. .. The digested DNA was then purified using a DNA clean and concentrator kit (Zymo Research, Irvine, CA).

    In Vitro:

    Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries
    Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB).

    Produced:

    Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet
    Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and T5 exonuclease (0.16 U/μL, New England Biolabs) were then added, and the reaction was incubated at 37 °C for 1 h to remove any pUCX2 without an insert.

    Fractionation:

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
    Article Snippet: For cytosolic HBV DNA in HepG2.2.15 cells, cells were treated with fractionation buffer (10 mM HEPES, 1.5 mM MgCl2 , 10 mM KCl) containing 1% NP-40. .. For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs.

    Staining:

    Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA
    Article Snippet: The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact. .. The products and remaining fragments of DNA were treated with T5 exonuclease (NEB) for 2 hr at 37 °C to degrade the background of unligated, linear ss DNA, leaving self-ligated ss DNA circles intact.

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    New England Biolabs t5 exonuclease
    Lesion-containing construct quality controls. (A) Schematic of the Fpg nicking assay. Fpg cleaves damages, such as 8-oxoG and 5-OHU, leaving a single-strand break, converting the construct from covalently closed (cc) to nicked form. (B) Lesion structures. (C) Representative images of Fpg and Nth nicked T5 exonuclease-treated lesion-containing and lesion-free control constructs. Fpg cleaves 8-oxoG, 5-OHU, and DHU, nicking the lesion-containing constructs almost entirely, but not the lesion-free controls, and Nth cleaves DHU.
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lesion-containing construct quality controls. (A) Schematic of the Fpg nicking assay. Fpg cleaves damages, such as 8-oxoG and 5-OHU, leaving a single-strand break, converting the construct from covalently closed (cc) to nicked form. (B) Lesion structures. (C) Representative images of Fpg and Nth nicked T5 exonuclease-treated lesion-containing and lesion-free control constructs. Fpg cleaves 8-oxoG, 5-OHU, and DHU, nicking the lesion-containing constructs almost entirely, but not the lesion-free controls, and Nth cleaves DHU.

    Journal: PLoS ONE

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    doi: 10.1371/journal.pone.0158581

    Figure Lengend Snippet: Lesion-containing construct quality controls. (A) Schematic of the Fpg nicking assay. Fpg cleaves damages, such as 8-oxoG and 5-OHU, leaving a single-strand break, converting the construct from covalently closed (cc) to nicked form. (B) Lesion structures. (C) Representative images of Fpg and Nth nicked T5 exonuclease-treated lesion-containing and lesion-free control constructs. Fpg cleaves 8-oxoG, 5-OHU, and DHU, nicking the lesion-containing constructs almost entirely, but not the lesion-free controls, and Nth cleaves DHU.

    Article Snippet: While we do not observe significant M13KO7 ssDNA contamination in purified constructs not treated with T5 exonuclease , treatment with T5 exonuclease can be employed if minimizing ssDNA contamination is preferred. (TIF) Click here for additional data file.

    Techniques: Construct

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT  T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG  constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT  construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Journal: PLoS ONE

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    doi: 10.1371/journal.pone.0158581

    Figure Lengend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Article Snippet: While we do not observe significant M13KO7 ssDNA contamination in purified constructs not treated with T5 exonuclease , treatment with T5 exonuclease can be employed if minimizing ssDNA contamination is preferred. (TIF) Click here for additional data file.

    Techniques: Construct, Purification, Negative Control, Positive Control

    Optimization for DNA integrity and mammalian transfection. (A) Schematic representing T5 exonuclease digestion of nicked, linear, and ssDNA. (B) Representative gel electrophoresis of a construct with and without T5 exonuclease treatment prior to purification and after purification. (C) Construct yields after T5 exonuclease treatment after initial purification (after) or directly in the second strand synthesis reaction (before), relative to non-T5 exonuclease treated construct (none). Error bars represent the standard deviation. (D) Live cell images of Ogg1 -/-  MEFs nucleofected with EGFP construct treated or not treated with T5 exonuclease or EGFP bacterial plasmid maxiprep, and stained with Hoechst 33342 dye. T5 exonuclease digestion of nicked and linear construct does not improve transfection efficiencies.

    Journal: PLoS ONE

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    doi: 10.1371/journal.pone.0158581

    Figure Lengend Snippet: Optimization for DNA integrity and mammalian transfection. (A) Schematic representing T5 exonuclease digestion of nicked, linear, and ssDNA. (B) Representative gel electrophoresis of a construct with and without T5 exonuclease treatment prior to purification and after purification. (C) Construct yields after T5 exonuclease treatment after initial purification (after) or directly in the second strand synthesis reaction (before), relative to non-T5 exonuclease treated construct (none). Error bars represent the standard deviation. (D) Live cell images of Ogg1 -/- MEFs nucleofected with EGFP construct treated or not treated with T5 exonuclease or EGFP bacterial plasmid maxiprep, and stained with Hoechst 33342 dye. T5 exonuclease digestion of nicked and linear construct does not improve transfection efficiencies.

    Article Snippet: While we do not observe significant M13KO7 ssDNA contamination in purified constructs not treated with T5 exonuclease , treatment with T5 exonuclease can be employed if minimizing ssDNA contamination is preferred. (TIF) Click here for additional data file.

    Techniques: Transfection, Nucleic Acid Electrophoresis, Construct, Purification, Standard Deviation, Plasmid Preparation, Staining

    Preparation of DNA template (A:C mismatch substrate) with A:C mismatch at a defined position.  a  Upper strand sequence containing A base and lower strand substrate containing original C base in pBS2/A:C are shown diagrammatically.  b  Experimental procedure for purification of pBS2/A:C.  c  Aliquots from various steps of the purification were analyzed on 0.8% agarose gel, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4,  Eco NI-treatment; lane 5, T5 exonuclease-treatment. Lower panel shows final purified DNA products (%). Open circular DNA (OC), linear DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrow.  d  Upper strand sequence containing A base in pBS2/A:C was sequenced. An A:C mismatch site is indicated by the arrow.  e  Lower strand sequence containing base A in pBS2/A:C was sequenced. The A:C mismatch site is indicated by the arrow

    Journal: Genes and Environment

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    doi: 10.1186/s41021-018-0112-5

    Figure Lengend Snippet: Preparation of DNA template (A:C mismatch substrate) with A:C mismatch at a defined position. a Upper strand sequence containing A base and lower strand substrate containing original C base in pBS2/A:C are shown diagrammatically. b Experimental procedure for purification of pBS2/A:C. c Aliquots from various steps of the purification were analyzed on 0.8% agarose gel, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment. Lower panel shows final purified DNA products (%). Open circular DNA (OC), linear DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrow. d Upper strand sequence containing A base in pBS2/A:C was sequenced. An A:C mismatch site is indicated by the arrow. e Lower strand sequence containing base A in pBS2/A:C was sequenced. The A:C mismatch site is indicated by the arrow

    Article Snippet: Nt.Bbv CI nicking endonuclease, Eco NI, Fpg, hOGG1, UDG, APE1, and T5 exonuclease were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Sequencing, Purification, Agarose Gel Electrophoresis, Staining, Countercurrent Chromatography

    a Experimental design. The plasmid pBS2-SDL was digested with a nicking endonuclease. An oligonucleotide containing a DNA lesion was hybridized with gap plasmid and ligated using T4 DNA ligase. Original plasmids in the sample are digested with restriction enzymes, except for DNA lesion bearing plasmids. T5 exonuclease cuts only the linear DNA plasmids digested by Eco NI, and does not work on sealed DNA plasmids containing a DNA lesion. b Covalently closed circular duplex DNA containing a single lesion. Sixty four-basepair oligonucleotides containing a single DNA lesion site within the Eco NI restriction enzyme site, two nicking endonuclease sites and the plasmid pBS2-SDL (2917 bp) are shown diagrammatically

    Journal: Genes and Environment

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    doi: 10.1186/s41021-018-0112-5

    Figure Lengend Snippet: a Experimental design. The plasmid pBS2-SDL was digested with a nicking endonuclease. An oligonucleotide containing a DNA lesion was hybridized with gap plasmid and ligated using T4 DNA ligase. Original plasmids in the sample are digested with restriction enzymes, except for DNA lesion bearing plasmids. T5 exonuclease cuts only the linear DNA plasmids digested by Eco NI, and does not work on sealed DNA plasmids containing a DNA lesion. b Covalently closed circular duplex DNA containing a single lesion. Sixty four-basepair oligonucleotides containing a single DNA lesion site within the Eco NI restriction enzyme site, two nicking endonuclease sites and the plasmid pBS2-SDL (2917 bp) are shown diagrammatically

    Article Snippet: Nt.Bbv CI nicking endonuclease, Eco NI, Fpg, hOGG1, UDG, APE1, and T5 exonuclease were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Plasmid Preparation

    One-pot synthesis of DNA repair substrate.  a  Experimental procedure for purification of pBS2/A:C omitting a column purification step.  b  Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4,  Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.

    Journal: Genes and Environment

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    doi: 10.1186/s41021-018-0112-5

    Figure Lengend Snippet: One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.

    Article Snippet: Nt.Bbv CI nicking endonuclease, Eco NI, Fpg, hOGG1, UDG, APE1, and T5 exonuclease were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Purification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Countercurrent Chromatography