t5 exonuclease  (New England Biolabs)


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  • 99
    Name:
    T5 Exonuclease
    Description:
    T5 Exonuclease 5 000 units
    Catalog Number:
    m0363l
    Price:
    265
    Size:
    5 000 units
    Category:
    Exonucleases
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    Structured Review

    New England Biolabs t5 exonuclease
    T5 Exonuclease
    T5 Exonuclease 5 000 units
    https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost"

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153158

    “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.
    Figure Legend Snippet: “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.

    Techniques Used: Produced, Plasmid Preparation, Transformation Assay, Clone Assay, Sequencing, Ligation

    2) 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

    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 "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

    5) 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

    6) 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

    7) Product Images from "Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication"

    Article Title: Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.01255-18

    Kinetics of HBcAg and HBV cccDNA synthesis upon induction with DMSO and hydrocortisone. HepG2 NTCP -P3 cells were infected with HBV at an MOI of 100 GC in the presence of 4% PEG 8000 for 12 h. HBV-infected cells were cultured in DME-F12 medium containing 3% FBS, 1% DMSO, and 5 μg/ml hydrocortisone. (A) At every day postinfection, the HBV-infected cells were lysed and the levels of HBcAg were determined by Western blotting. (B) HBV cccDNA in the cells was subjected to Hirt extraction, followed by digestion with T5 exonuclease and quantification by qPCR using cccDNA-specific primers and probe. Average values (±SD) derived from three experiments are plotted.
    Figure Legend Snippet: Kinetics of HBcAg and HBV cccDNA synthesis upon induction with DMSO and hydrocortisone. HepG2 NTCP -P3 cells were infected with HBV at an MOI of 100 GC in the presence of 4% PEG 8000 for 12 h. HBV-infected cells were cultured in DME-F12 medium containing 3% FBS, 1% DMSO, and 5 μg/ml hydrocortisone. (A) At every day postinfection, the HBV-infected cells were lysed and the levels of HBcAg were determined by Western blotting. (B) HBV cccDNA in the cells was subjected to Hirt extraction, followed by digestion with T5 exonuclease and quantification by qPCR using cccDNA-specific primers and probe. Average values (±SD) derived from three experiments are plotted.

    Techniques Used: Infection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Derivative Assay

    8) 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

    9) 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

    10) 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

    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 "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

    13) 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

    14) 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

    15) 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

    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 "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

    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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) Product Images from "Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning"

    Article Title: Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2019.00460

    Nimble Cloning system.  (A)  Schematic of the destination and entry vectors in the Nimble Cloning system. The destination vector contains the NC frame comprising the “adapter 1– Sfi I– ccdB  gene– Sfi I–adapter 2” sequence, whereas the entry vector contains the “ Sfi I–adapter 1–XcmI– ccdB  gene–XcmI–adapter 2– Sfi I” sequence at the cloning site.  (B)  Schematic of the Nimble Cloning method. The PCR product flanked by the adapters or the DNA fragment in the entry clone can be cloned into the circular destination vector in a one-step Nimble Cloning reaction. The DNA fragment can be inserted into the entry vector to form the entry clone via TA cloning, or by Gibson cloning using the adapters as overlapping sequences, after the entry vector is digested with XcmI. Nimble Cloning involves  Sfi I and T5 exonuclease.
    Figure Legend Snippet: Nimble Cloning system. (A) Schematic of the destination and entry vectors in the Nimble Cloning system. The destination vector contains the NC frame comprising the “adapter 1– Sfi I– ccdB gene– Sfi I–adapter 2” sequence, whereas the entry vector contains the “ Sfi I–adapter 1–XcmI– ccdB gene–XcmI–adapter 2– Sfi I” sequence at the cloning site. (B) Schematic of the Nimble Cloning method. The PCR product flanked by the adapters or the DNA fragment in the entry clone can be cloned into the circular destination vector in a one-step Nimble Cloning reaction. The DNA fragment can be inserted into the entry vector to form the entry clone via TA cloning, or by Gibson cloning using the adapters as overlapping sequences, after the entry vector is digested with XcmI. Nimble Cloning involves Sfi I and T5 exonuclease.

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, TA Cloning

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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

    38) 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

    39) 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

    40) 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

    41) 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

    42) 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

    43) 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

    44) Product Images from "Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching"

    Article Title: Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching

    Journal: ACS Omega

    doi: 10.1021/acsomega.9b02676

    (A) Effects of different enzymes on the production of rx and sc pAB1_FL905. rx (lanes 1–4) and sc (lanes 5–8) pAB1_FL905 were generated according to   Figure   1  and also as described in “  Materials and Methods .” The DNA molecules were isolated and subjected to 1% agarose gel electrophoresis in 1× TAE buffer. Symbols and abbreviations: T4 DNAL, T4 DNA ligase; T4 DNAP, T4 DNA polymerase; T5 exo, T5 exonuclease; nk, nicked plasmid; sc, supercoiled plasmid; rx, relaxed plasmids; FL905, oligomer FL905. Lane 9 is the NEB 1 kb DNA ladder. (B) Fluorescence spectra of sc (σ = −0.06; dotted line) and rx (σ = 0; solid line) of pAB1_FL924 with λ ex  = 532 nm. (C) Fluorescence intensity of pAB1_FL924 is dependent of supercoiling density (σ). DNA topoisomeras with a mean ΔLk were generated as described under   Materials and Methods  and used here. The fluorescence intensity of the same concentration of pAB1_FL924 samples was measured using a microplate reader with λ ex  = 550 nm and λ em  = 580 nm. (D,E) Analysis of DNA topoisomers using 1% agarose gel electrophoreses in the absence (D) and presence of 2.5 μg/mL chloroquine (E) to determine the supercoiling density of different pAB1_FL924 samples. Lanes 1 to 9 are DNA samples relaxed by variola DNA topoisomerase I in the presence of 0, 0.5, 1, 1.5, 2, 2.5, 3.75, 5, and 7.5 μM of EB, respectively. Lanes 10 and 11 are supercoiled and relaxed pAB1, respectively.
    Figure Legend Snippet: (A) Effects of different enzymes on the production of rx and sc pAB1_FL905. rx (lanes 1–4) and sc (lanes 5–8) pAB1_FL905 were generated according to Figure 1 and also as described in “ Materials and Methods .” The DNA molecules were isolated and subjected to 1% agarose gel electrophoresis in 1× TAE buffer. Symbols and abbreviations: T4 DNAL, T4 DNA ligase; T4 DNAP, T4 DNA polymerase; T5 exo, T5 exonuclease; nk, nicked plasmid; sc, supercoiled plasmid; rx, relaxed plasmids; FL905, oligomer FL905. Lane 9 is the NEB 1 kb DNA ladder. (B) Fluorescence spectra of sc (σ = −0.06; dotted line) and rx (σ = 0; solid line) of pAB1_FL924 with λ ex = 532 nm. (C) Fluorescence intensity of pAB1_FL924 is dependent of supercoiling density (σ). DNA topoisomeras with a mean ΔLk were generated as described under Materials and Methods and used here. The fluorescence intensity of the same concentration of pAB1_FL924 samples was measured using a microplate reader with λ ex = 550 nm and λ em = 580 nm. (D,E) Analysis of DNA topoisomers using 1% agarose gel electrophoreses in the absence (D) and presence of 2.5 μg/mL chloroquine (E) to determine the supercoiling density of different pAB1_FL924 samples. Lanes 1 to 9 are DNA samples relaxed by variola DNA topoisomerase I in the presence of 0, 0.5, 1, 1.5, 2, 2.5, 3.75, 5, and 7.5 μM of EB, respectively. Lanes 10 and 11 are supercoiled and relaxed pAB1, respectively.

    Techniques Used: Generated, Isolation, Agarose Gel Electrophoresis, Plasmid Preparation, Fluorescence, Concentration Assay

    Related Articles

    DNA Extraction:

    Article Title: Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication
    Article Snippet: HBV DNA was extracted from the cells or from the medium using DNA isolation kits from Qiagen according to the manufacturer’s protocol. .. HBV cccDNA in the cells was extracted with the Hirt method ( ) and was further treated with T5 exonuclease (New England BioLabs), which degrades DNAs with open 5ʹ and 3ʹ ends, such as HBV rcDNA, similarly to exonucleases Exo I and III ( ).

    Clone Assay:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: .. T5 exonuclease in a Tris buffer with PEG 8000 works well for cloning The Gibson method applies three enzymes, and it can be simplified by removing Taq DNA ligase without reducing the cloning efficiency ( , ). .. To test whether the method could be further simplified, we checked the requirement of the enzymes and other components in the Gibson system for cloning Pkat-eGFP into pBluescript SK- (Figure ).

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: The gfp gene was amplified from the plasmid pGreenTIR by PCR using the primers gfp -F and gfp -R and cloned into the Nsi I/Xho I restricted vector pLC, generating the reporter plasmid pLG. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost
    Article Snippet: Gibson assembly The same insert and PCR-linearized plasmid preparations as for co-transformation cloning were used. .. Each Gibson assembly reaction consisted of 2.7 μl 5x IT buffer, 2 μl insert-plasmid mastermix (containing 75 ng plasmid and an 8-fold molar excess of insert), 5.3 μl 1:1000 diluted T5 exonuclease (New England Biolabs M0363S, 10’000 U/ml), 1.6 μl of 1:10 diluted Phusion HF DNA polymerase (NEB M0530L, 2’000 U/ml), 1.3 μl Taq DNA ligase (NEB M0208L, 40’000 U/ml, undiluted) and H2 0 to a final volume of 13.5 μl.

    RNA Extraction:

    Article Title: Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication
    Article Snippet: Paragraph title: DNA and RNA extraction. ... HBV cccDNA in the cells was extracted with the Hirt method ( ) and was further treated with T5 exonuclease (New England BioLabs), which degrades DNAs with open 5ʹ and 3ʹ ends, such as HBV rcDNA, similarly to exonucleases Exo I and III ( ).

    Amplification:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: In brief, the DNA fragment with five tandem repeats of the core-tac -promoter with flanked extensions was amplified by PCR using the primers P5tac -F and P5tac -R. Fragment 1 was obtained by digesting the resulting PCR products with BamH I, and fragment 2 was generated by cutting with Hind III and BamH I. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Agarose Gel Electrophoresis:

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
    Article Snippet: Hirt DNA was separated on an 1.2% agarose gel and transferred onto a positively charged nylon membrane via upward capillary transfer. .. To confirm the presence of cccDNA, Hirt extracted DNA was digested with T5 exonuclease (New England Biolabs) at 37 °C for 30 min.

    In Vitro:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min. .. The resulting constructs containing different promoters were then transformed into competent cells and were firstly screened based on the fluorescence signal and PCR detection.

    Fluorescence:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min. .. The resulting constructs containing different promoters were then transformed into competent cells and were firstly screened based on the fluorescence signal and PCR detection.

    Synthesized:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: The 5CPtac s promoter cluster was synthesized in the form of five core-tac -promoters in tandem and inserted into Hind III and BamH I sites of the plasmid pLG to produce the plasmid p5TG. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Southern Blot:

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
    Article Snippet: Paragraph title: Southern blot analysis ... To confirm the presence of cccDNA, Hirt extracted DNA was digested with T5 exonuclease (New England Biolabs) at 37 °C for 30 min.

    Generated:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: In brief, the DNA fragment with five tandem repeats of the core-tac -promoter with flanked extensions was amplified by PCR using the primers P5tac -F and P5tac -R. Fragment 1 was obtained by digesting the resulting PCR products with BamH I, and fragment 2 was generated by cutting with Hind III and BamH I. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Construct:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: A one-step method of assembling several overlapping DNA fragments was adopted to construct the different MCPtac s promoter clusters [ ]. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    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: .. 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. ..

    Purification:

    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: .. 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. ..

    Polymerase Chain Reaction:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: In brief, the DNA fragment with five tandem repeats of the core-tac -promoter with flanked extensions was amplified by PCR using the primers P5tac -F and P5tac -R. Fragment 1 was obtained by digesting the resulting PCR products with BamH I, and fragment 2 was generated by cutting with Hind III and BamH I. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost
    Article Snippet: Gibson assembly The same insert and PCR-linearized plasmid preparations as for co-transformation cloning were used. .. Each Gibson assembly reaction consisted of 2.7 μl 5x IT buffer, 2 μl insert-plasmid mastermix (containing 75 ng plasmid and an 8-fold molar excess of insert), 5.3 μl 1:1000 diluted T5 exonuclease (New England Biolabs M0363S, 10’000 U/ml), 1.6 μl of 1:10 diluted Phusion HF DNA polymerase (NEB M0530L, 2’000 U/ml), 1.3 μl Taq DNA ligase (NEB M0208L, 40’000 U/ml, undiluted) and H2 0 to a final volume of 13.5 μl.

    Incubation:

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost
    Article Snippet: Each Gibson assembly reaction consisted of 2.7 μl 5x IT buffer, 2 μl insert-plasmid mastermix (containing 75 ng plasmid and an 8-fold molar excess of insert), 5.3 μl 1:1000 diluted T5 exonuclease (New England Biolabs M0363S, 10’000 U/ml), 1.6 μl of 1:10 diluted Phusion HF DNA polymerase (NEB M0530L, 2’000 U/ml), 1.3 μl Taq DNA ligase (NEB M0208L, 40’000 U/ml, undiluted) and H2 0 to a final volume of 13.5 μl. .. The reactions were incubated in the PCR machine at 50°C for one hour with the hotlid at 80°C.

    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.

    Expressing:

    Article Title: Cellular reagents for diagnostics and synthetic biology
    Article Snippet: .. 2x108 Top10 lyophilized cellular reagents expressing Taq DNA polymerase, T5 exonuclease, or Taq DNA ligase were rehydrated using 30 μl of water. ..

    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). .. For Gibson assemblies using cellular reagents the pure enzymes were substituted with individual Top10 E . coli cellular reagents expressing Taq DNA Ligase, Taq DNA polymerase, and T5 exonuclease.

    Article Title: Cellular reagents for diagnostics and synthetic biology
    Article Snippet: .. For Gibson assemblies using cellular reagents the pure enzymes were substituted with individual Top10 E . coli cellular reagents expressing Taq DNA Ligase, Taq DNA polymerase, and T5 exonuclease. ..

    Modification:

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
    Article Snippet: Southern blot analysis DNA was extracted from HBV-infected rhesus macaque PH using a modified Hirt extraction, as previously described . .. To confirm the presence of cccDNA, Hirt extracted DNA was digested with T5 exonuclease (New England Biolabs) at 37 °C for 30 min.

    Planar Chromatography:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: The gfp gene was amplified from the plasmid pGreenTIR by PCR using the primers gfp -F and gfp -R and cloned into the Nsi I/Xho I restricted vector pLC, generating the reporter plasmid pLG. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Transformation Assay:

    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).

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min. .. The resulting constructs containing different promoters were then transformed into competent cells and were firstly screened based on the fluorescence signal and PCR detection.

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost
    Article Snippet: Each Gibson assembly reaction consisted of 2.7 μl 5x IT buffer, 2 μl insert-plasmid mastermix (containing 75 ng plasmid and an 8-fold molar excess of insert), 5.3 μl 1:1000 diluted T5 exonuclease (New England Biolabs M0363S, 10’000 U/ml), 1.6 μl of 1:10 diluted Phusion HF DNA polymerase (NEB M0530L, 2’000 U/ml), 1.3 μl Taq DNA ligase (NEB M0208L, 40’000 U/ml, undiluted) and H2 0 to a final volume of 13.5 μl. .. After cooling down to 4°C, 1/20 (3.75 ng plasmid and an 8-fold molar excess of insert) of each of the mixtures was transferred to 100 μl aliquots of chemically competent E . coli cells and incubated on ice for 15 minutes, followed by heat shock transformation.

    Plasmid Preparation:

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli
    Article Snippet: The plasmid p5TG was linearized by Hind III to produce fragment 3. .. Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost
    Article Snippet: .. Each Gibson assembly reaction consisted of 2.7 μl 5x IT buffer, 2 μl insert-plasmid mastermix (containing 75 ng plasmid and an 8-fold molar excess of insert), 5.3 μl 1:1000 diluted T5 exonuclease (New England Biolabs M0363S, 10’000 U/ml), 1.6 μl of 1:10 diluted Phusion HF DNA polymerase (NEB M0530L, 2’000 U/ml), 1.3 μl Taq DNA ligase (NEB M0208L, 40’000 U/ml, undiluted) and H2 0 to a final volume of 13.5 μl. ..

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    New England Biolabs t5 exonuclease
    “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with <t>T5</t> exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.

    Journal: PLoS ONE

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

    doi: 10.1371/journal.pone.0153158

    Figure Lengend Snippet: “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.

    Article Snippet: Each Gibson assembly reaction consisted of 2.7 μl 5x IT buffer, 2 μl insert-plasmid mastermix (containing 75 ng plasmid and an 8-fold molar excess of insert), 5.3 μl 1:1000 diluted T5 exonuclease (New England Biolabs M0363S, 10’000 U/ml), 1.6 μl of 1:10 diluted Phusion HF DNA polymerase (NEB M0530L, 2’000 U/ml), 1.3 μl Taq DNA ligase (NEB M0208L, 40’000 U/ml, undiluted) and H2 0 to a final volume of 13.5 μl.

    Techniques: Produced, Plasmid Preparation, Transformation Assay, Clone Assay, Sequencing, Ligation

    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.

    Journal: PLoS ONE

    Article Title: Cellular reagents for diagnostics and synthetic biology

    doi: 10.1371/journal.pone.0201681

    Figure Lengend 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.

    Article Snippet: For Gibson assemblies using cellular reagents the pure enzymes were substituted with individual Top10 E . coli cellular reagents expressing Taq DNA Ligase, Taq DNA polymerase, and T5 exonuclease.

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

    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.

    Journal: PLoS ONE

    Article Title: Cellular reagents for diagnostics and synthetic biology

    doi: 10.1371/journal.pone.0201681

    Figure Lengend 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.

    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).

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