t5 exonuclease (New England Biolabs)
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Name:
T5 Exonuclease
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Catalog Number:
M0363L
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Score:
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Structured Review
New England Biolabs
t5 exonuclease
https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
Average 99 stars, based on 11 article reviews
Price from $9.99 to $1999.99
https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
Average 99 stars, based on 11 article reviews
Price from $9.99 to $1999.99
t5 exonuclease - by Bioz Stars,
2019-12
99/100 stars
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Images
1) Product Images from "Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques"
Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
Journal: Nature Communications
doi: 10.1038/s41467-017-01953-y
Figure Legend Snippet: In vitro HBV infection of rhesus macaque PH. a Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP. b Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488. c Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N = 1). Figure is representative data of two separate experiments. d HBV DNA qPCR on the same supernatants shown in c . Each condition represents a single-biological sample ( N = 1). e Total intracellular DNA from 1 × 10 6 rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates. f Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments. g Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4 or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N = 1)
Techniques Used: In Vitro, Infection, Labeling, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Southern Blot, Purification, Marker
2) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
3) Product Images from "Cellular reagents for diagnostics and synthetic biology"
Article Title: Cellular reagents for diagnostics and synthetic biology
Journal: PLoS ONE
doi: 10.1371/journal.pone.0201681
Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay
4) Product Images from "Patch cloning method for multiple site-directed and saturation mutagenesis"
Article Title: Patch cloning method for multiple site-directed and saturation mutagenesis
Journal: BMC Biotechnology
doi: 10.1186/1472-6750-13-91
Figure Legend Snippet: Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation
5) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"
Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site
Journal: Genes and Environment
doi: 10.1186/s41021-018-0112-5
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
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
Figure Legend Snippet: One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.
Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Countercurrent Chromatography
6) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
7) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
8) Product Images from "Cellular reagents for diagnostics and synthetic biology"
Article Title: Cellular reagents for diagnostics and synthetic biology
Journal: PLoS ONE
doi: 10.1371/journal.pone.0201681
Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay
9) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
10) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
11) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
12) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"
Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
Journal: PLoS ONE
doi: 10.1371/journal.pone.0158581
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
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
Figure Legend Snippet: Optimization for DNA integrity and mammalian transfection. (A) Schematic representing T5 exonuclease digestion of nicked, linear, and ssDNA. (B) Representative gel electrophoresis of a construct with and without T5 exonuclease treatment prior to purification and after purification. (C) Construct yields after T5 exonuclease treatment after initial purification (after) or directly in the second strand synthesis reaction (before), relative to non-T5 exonuclease treated construct (none). Error bars represent the standard deviation. (D) Live cell images of Ogg1 -/- MEFs nucleofected with EGFP construct treated or not treated with T5 exonuclease or EGFP bacterial plasmid maxiprep, and stained with Hoechst 33342 dye. T5 exonuclease digestion of nicked and linear construct does not improve transfection efficiencies.
Techniques Used: Transfection, Nucleic Acid Electrophoresis, Construct, Purification, Standard Deviation, Plasmid Preparation, Staining
13) Product Images from "QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides"
Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides
Journal: PLoS ONE
doi: 10.1371/journal.pone.0175146
Figure Legend Snippet: Circularization of linear plasmid library and removal of starting matrix. (a) Time course of the circularization reaction: a mix of four enzymes (T5 exonuclease, DNA polymerase, DNA ligase and DpnI) was added to the amplified linear plasmids and incubated for one hour at 50 C. The amount of T5 exonuclease was reduced 4-fold compared to Gibson’s protocol. Circularization products (left side) are also analyzed by restriction with AvaI (right side). The band at 3.6 kb (black arrow) is only present when the plasmids are sealed. (b) DpnI is cleaving the starting matrix at 50 C while the synthetic PCR product is resistant to its action.
Techniques Used: Plasmid Preparation, Amplification, Incubation, Polymerase Chain Reaction
14) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
15) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
16) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay
17) Product Images from "Unique nucleotide sequence (UNS)-guided assembly of repetitive DNA parts for synthetic biology applications"
Article Title: Unique nucleotide sequence (UNS)-guided assembly of repetitive DNA parts for synthetic biology applications
Journal:
doi: 10.1038/nprot.2014.145
Figure Legend Snippet: Sequence considerations when using standard part vectors When using currently available part vectors , restriction sites lying just outside of the UNSs are digested to generate linear parts. Shown is an example in which pFLU1U2 containing “Part A” is digested with AscI and MauBI. This digestion leaves behind terminal nucleotides from the restriction sites (orange). The 5′ nucleotides at the termini are digested by T5 exonuclease during isothermal assembly, but those at the 3′ ends are left behind. When Part A anneals to another part, “Part B” (bottom), the terminal nucleotides in Part A anneal with nucleotides internal to the UNSs in Part B and vice versa . Caution must be taken to ensure these terminal nucleotides do not create mismatches that might interfere with assembly.
Techniques Used: Sequencing
18) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"
Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky1169
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
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
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
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
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
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
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
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
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
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The Hirt-extracted DNA was purified and treated with Incubation:Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet Article Snippet: For pINF assembly, pUCX2 (1 μg) and insert DNA were combined at a 1:4 molar ratio in a 80 μL reaction with ATP (1 mM), T4 DNA ligase (6.65 U/μL, New England Biolabs), BsaI-HF (0.66 U/μL, New England Biolabs), and CutSmart Buffer (1×, New England Biolabs) and subjected to the following temperature regime: [37 °C 20 min | 40 × (37 °C 5 min | 16 °C 10 min | 22 °C 5 min) | 37 °C 20 min | 50 °C 15 min | 70 °C 30 min]. .. BsaI-HF (0.33 U/μL) and Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: The resulting single-stranded (ss) fragments were incubated in 50 mM Tris-HCl, pH 7.5, 40 mM NaCl, 5 mM MgCl2 , 1 mM DTT, 30 µM ATP (Sigma-Aldrich), and 1 µM AtRNL for 1 hr at 30 °C, followed by purification. .. The products and remaining fragments of DNA were treated with Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle Article Snippet: Briefly, cells were trypsinized and re-suspended in provided T1 buffer and incubated with proteinase K and B3 buffer at 70 °C for 1 hour and following steps were stick to the protocol. .. For cccDNA quantification, DNA samples were digested with Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries Article Snippet: In short, the library was amplified using gene specific Gibson adaptor primers ( ) bearing an 18 base pair extension that overlaps with the RTS upstream and RTS downstream fragments. .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U Introduce:Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Primers Fsp, Rsp-1, and Rsp-2 were used to introduce a terminator at the end of the spectinomycin resistance gene and primers Fhfq-1 and Fhfq-2 were used to introduce the 3xFLAG. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Expressing:Article Title: Cellular reagents for diagnostics and synthetic biology Article Snippet: Lyophilized cellular reagents were prepared as follows for use in Gibson assemblies. .. 2x108 Top10 lyophilized cellular reagents expressing Taq DNA polymerase, Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis Article Snippet: In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with Article Title: Cellular reagents for diagnostics and synthetic biology Article Snippet: Assemblies using pure enzymes contained 0.08 units of Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle Article Snippet: For cccDNA quantification, DNA samples were digested with Knock-In:Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet Article Snippet: BsaI-HF (0.33 U/μL) and Transformation Assay:Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis Article Snippet: The resulting plasmid was verified by sequencing and transformed into B. subtilis competent cells, resulting in strain UG10. recA and yneA promoter reporters were constructed by amplifying the promoter regions with the LB141-LB142 and LB145-LB146 primer pairs, respectively, and using the genomic DNA of strain 168 as the template. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with 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 Gel Purification:Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Transfection:Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells Article Snippet: Higher yields can be obtained if the T5 exonuclease treatment is performed directly in the second strand synthesis reaction ( ). .. We find that treatment with Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using Southern Blot:Article Title: The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection Article Snippet: Paragraph title: Southern blotting ... A total of 1 μg of the extracted DNA was digested with 10 units of Ligation:Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U Infection:Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle Article Snippet: For analysis of intracellular total HBV DNA and cccDNA from infected HepG2-NTCP cells, DNA from total cell lysates was prepared using the NucleoSpin Tissue Kit (Macherey-Nagel 740952.250) according to manufacturer’s protocol with minor modifications. .. For cccDNA quantification, DNA samples were digested with other:Article Title: Cellular reagents for diagnostics and synthetic biology Article Snippet: To most simply carry out Gibson assembly with cellular reagents we merely lyophilized three cell lines that expressed Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease, respectively. Article Title: Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning Article Snippet: All the enzymes including type II restriction enzymes (EcoRI, BamHI, SalI and Bau36I), type IIS restriction enzymes (BsaI, BbsI and BsmBI), T4 DNA ligase, Phusion enzyme, and Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: After dilution, the resulting products were treated with T5 exonuclease for 2 hr at 37 °C. Sequencing:Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis Article Snippet: Overlapping sequences (20 nucleotides [nt]) were inserted into the primers to the adjacent sequences of interest for cloning using the one-step isothermal assembly method ( ). .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: The products and remaining fragments of DNA were treated with Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U DNA Extraction:Article Title: Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes Article Snippet: Quantitative PCR for HBV were performed using HBV specific primers and normalized to PRNP housekeeping gene as previously described . .. After total DNA isolation and Nucleic Acid Electrophoresis:Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Real-time Polymerase Chain Reaction:Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus Article Snippet: Paragraph title: qPCR and RT-qPCR ... The Hirt-extracted DNA was purified and treated with Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle Article Snippet: Paragraph title: Preparation of viral DNA and quantitative PCR analysis ... For cccDNA quantification, DNA samples were digested with Mutagenesis:Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis Article Snippet: In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Paragraph title: Construction of hfq mutant strains ... Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U Isolation:Article Title: The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection Article Snippet: DNA was isolated from the nuclei of HBV-infected PHH by Hirt extraction as previously described [ ]. .. A total of 1 μg of the extracted DNA was digested with 10 units of Polymerase Chain Reaction:Article Title: Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases Article Snippet: Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis Article Snippet: The PCR product and the amyE integration vector pSG1729 ( ) were digested with XhoI and EcoRI restriction enzymes and ligated. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: The products and remaining fragments of DNA were treated with Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: The upstream homologous arm was PCR amplified using primers Fup and Rup, and the downstream homologous arm amplified by Fhfq-1, Fhfq-2, and Rhfq. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U Purification:Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet Article Snippet: The resulting UBP containing dsDNA was purified using a DNA Clean & Concentrator-5 (Zymo Research) according to manufacturer recommendations. .. BsaI-HF (0.33 U/μL) and Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides Article Snippet: Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs. .. Phusion DNA polymerase, DpnI, Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus Article Snippet: The cccDNA purification was performed using a modified Hirt extraction procedure [ ]. .. The Hirt-extracted DNA was purified and treated with Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: The resulting single-stranded (ss) fragments were incubated in 50 mM Tris-HCl, pH 7.5, 40 mM NaCl, 5 mM MgCl2 , 1 mM DTT, 30 µM ATP (Sigma-Aldrich), and 1 µM AtRNL for 1 hr at 30 °C, followed by purification. .. The products and remaining fragments of DNA were treated with Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries Article Snippet: In short, the library was amplified using gene specific Gibson adaptor primers ( ) bearing an 18 base pair extension that overlaps with the RTS upstream and RTS downstream fragments. .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U Reverse Transcription Polymerase Chain Reaction:Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques Article Snippet: Total HBV DNA and RNA were quantified using the TaqMan Fast Advanced Master Mix (Applied Biosystems) and AgPath-ID One-Step RT-PCR kit (Life Technologies), respectively. .. DNA was eluted from NucleoSpin Tissue Columns with 100 μl pre-warmed (70 °C) buffer and subjected to Quantitative RT-PCR:Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus Article Snippet: Paragraph title: qPCR and RT-qPCR ... The Hirt-extracted DNA was purified and treated with Polyacrylamide Gel Electrophoresis:Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: The products and remaining fragments of DNA were treated with Plasmid Preparation:Article Title: Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases Article Snippet: 10G Hi-Control and BL21(DE3) Hi-Control E.coli were obtained from an Expresso T7 cloning kit (Lucigen). pET24a vector was purchased from EMD Millipore. .. Article Title: The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis Article Snippet: The amyE integration vector pPG40 ( ) containing the β-galactosidase gene ( lacZ ) was amplified with the LB139-LB140 and LB143-LB144 primer pairs for cloning PrecA and PyneA, respectively. .. In short, equimolar concentrations of the two DNA fragments sharing terminal sequence overlaps (20 nt) were mixed with Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle Article Snippet: Absolute copy numbers were calculated referring to a standard based on the linearized HBV coding plasmid pJO19. .. For cccDNA quantification, DNA samples were digested with Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using Article Title: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts Article Snippet: Briefly, primers were designed with 15-nt overhangs of desired upstream or downstream sequences and used to amplify the pieces for the assembly and purified from the primer dimers by gel electrophoresis followed by gel purification. .. Fragments and the pUC57 vector were mixed in a molar ratio of 3:1 (about 120 ng total DNA) and added to 0.5 U Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques Article Snippet: Quantification was assessed relative to an absolute standard curve using the plasmid pCEP4 with the targeted insert as template. .. DNA was eluted from NucleoSpin Tissue Columns with 100 μl pre-warmed (70 °C) buffer and subjected to Electrophoresis:Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides Article Snippet: Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs. .. Phusion DNA polymerase, DpnI, Agarose Gel Electrophoresis:Article Title: The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection Article Snippet: A total of 1 μg of the extracted DNA was digested with 10 units of In Vitro:Article Title: An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries Article Snippet: Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U T5 exonuclease (Epicenter), 0.5 U Phusion polymerase (NEB), and 80 U Taq ligase (NEB). .. Assemblies were performed as follows, gel purified library (100ng) was incubated with a 2-fold molar excess of the upstream and downstream regulatory regions wre incubated a 20 ul reaction containing 100 mM Tris pH 7.5, 10 mM MgCl2 , 0.4 mM each of dGTP, dCTP, dATP, dTTP, 10 mM DTT, 50 mg/ml PEG-8000, 1 mM NAD, 0.08 U Produced:Article Title: Reprograming the replisome of a semi-synthetic organism for the expansion of the genetic alphabet Article Snippet: UBP containing dsDNA was produced with a 50-μL PCR with chemically synthesized UBP containing oligonucleotides (0.025 ng/μL), primers introducing BsaI sites and vector homology (1 μM, ), d TPT3 TP (100 μM), d NaM TP (100 μM), dNTPs (200 μM), MgSO4 (1.2 mM), One Taq DNA Polymerase (0.025 U/μL), and One Taq Standard Reaction Buffer (1×, New England Biolabs). .. BsaI-HF (0.33 U/μL) and Fractionation:Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle Article Snippet: For cytosolic HBV DNA in HepG2.2.15 cells, cells were treated with fractionation buffer (10 mM HEPES, 1.5 mM MgCl2 , 10 mM KCl) containing 1% NP-40. .. For cccDNA quantification, DNA samples were digested with Staining:Article Title: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA Article Snippet: The products and remaining fragments of DNA were treated with |