Structured Review

Promega t4 ligase
Time course of BER. 35-μl reactions were performed as described under “Experimental Procedures” with UL2, APE, and UL30 in the presence of either <t>T4</t> ligase (0.001 units/μl) ( A ), ligase I ( B ), or ligase IIIα-XRCC1 ( C ). 5-μl aliquots were removed at the times indicated. Activity is expressed as the fraction of maximum for the nicked ( N ) (●) and ligation ( L ) (○) products. The insets in each panel show the relevant gel images.
T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/t4 ligase/product/Promega
Average 96 stars, based on 102 article reviews
Price from $9.99 to $1999.99
t4 ligase - by Bioz Stars, 2020-01
96/100 stars

Images

1) Product Images from "Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *"

Article Title: Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.010413

Time course of BER. 35-μl reactions were performed as described under “Experimental Procedures” with UL2, APE, and UL30 in the presence of either T4 ligase (0.001 units/μl) ( A ), ligase I ( B ), or ligase IIIα-XRCC1 ( C ). 5-μl aliquots were removed at the times indicated. Activity is expressed as the fraction of maximum for the nicked ( N ) (●) and ligation ( L ) (○) products. The insets in each panel show the relevant gel images.
Figure Legend Snippet: Time course of BER. 35-μl reactions were performed as described under “Experimental Procedures” with UL2, APE, and UL30 in the presence of either T4 ligase (0.001 units/μl) ( A ), ligase I ( B ), or ligase IIIα-XRCC1 ( C ). 5-μl aliquots were removed at the times indicated. Activity is expressed as the fraction of maximum for the nicked ( N ) (●) and ligation ( L ) (○) products. The insets in each panel show the relevant gel images.

Techniques Used: Activity Assay, Ligation

Completion of BER; formation of ligation product. Reactions were performed as described under “Experimental Procedures” with the indicated proteins. Storage phosphorimage of reaction products obtained with UL2, APE, and UL30 ( lane 1 ) and identical reactions supplemented with T4 ligase (1.5 units) ( lane 2 ), ligase I ( lane 3 ), or ligase IIIα-XRCC1 ( lane 4 ) are shown. Lane 5 , DNA only; lane 6 , linear 100-mer. The positions of nicked product ( N ), ligated product ( L ), and of the 100-mer are as indicated. The values in italics below the lane numbers indicate the L/N ratios.
Figure Legend Snippet: Completion of BER; formation of ligation product. Reactions were performed as described under “Experimental Procedures” with the indicated proteins. Storage phosphorimage of reaction products obtained with UL2, APE, and UL30 ( lane 1 ) and identical reactions supplemented with T4 ligase (1.5 units) ( lane 2 ), ligase I ( lane 3 ), or ligase IIIα-XRCC1 ( lane 4 ) are shown. Lane 5 , DNA only; lane 6 , linear 100-mer. The positions of nicked product ( N ), ligated product ( L ), and of the 100-mer are as indicated. The values in italics below the lane numbers indicate the L/N ratios.

Techniques Used: Ligation

Product formation is dependent on UL2, APE, UL30, and ligase. Storage phosphorimages of reactions performed as described under “Experimental Procedures” with the indicated components. Lane 1 , UL2; lane 2 , APE; lane 3 ; UL30; lane 4 , T4 ligase (1.5 units); lane 5 , ligase I; lane 6 , ligase IIIα-XRCC1; lane 7 , UL2 and APE; lane 8 , APE and UL30; lane 9 , UL2 and UL30; lane 10 , UL2, APE and UL30; lane 11 , UL2, APE, UL30 and T4 ligase (1.5 units); lane 12 , UL2, APE, UL30, and ligase I; lane 13 , UL2, APE, UL30, and ligase IIIα-XRCC1; lane 14 , DNA only. The positions of nicked ( N ) and ligated ( L ) products are as indicated.
Figure Legend Snippet: Product formation is dependent on UL2, APE, UL30, and ligase. Storage phosphorimages of reactions performed as described under “Experimental Procedures” with the indicated components. Lane 1 , UL2; lane 2 , APE; lane 3 ; UL30; lane 4 , T4 ligase (1.5 units); lane 5 , ligase I; lane 6 , ligase IIIα-XRCC1; lane 7 , UL2 and APE; lane 8 , APE and UL30; lane 9 , UL2 and UL30; lane 10 , UL2, APE and UL30; lane 11 , UL2, APE, UL30 and T4 ligase (1.5 units); lane 12 , UL2, APE, UL30, and ligase I; lane 13 , UL2, APE, UL30, and ligase IIIα-XRCC1; lane 14 , DNA only. The positions of nicked ( N ) and ligated ( L ) products are as indicated.

Techniques Used:

2) Product Images from "Enzyme-guided DNA Sewing Architecture"

Article Title: Enzyme-guided DNA Sewing Architecture

Journal: Scientific Reports

doi: 10.1038/srep17722

Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.
Figure Legend Snippet: Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.

Techniques Used: Ligation, Activity Assay, Sequencing, Concentration Assay

3) Product Images from "Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase"

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

Journal: Journal of Biological Engineering

doi: 10.1186/1754-1611-1-7

Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
Figure Legend Snippet: Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

Techniques Used: Subcloning, Plasmid Preparation, Activity Assay, Marker

Related Articles

Clone Assay:

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: Paragraph title: Plasmid DNA Cloning ... The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1.

Article Title: cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression *
Article Snippet: PCR was used to amplify mouse Cd39 promoter-luciferase reporter constructs representing 5′-deleted Cd39 upstream sequences, and these PCR products were subcloned into the pGL3-basic vector (Promega) using a T4 ligase and Quick Ligasing buffer (Promega). .. The Cd39 promoter −220 to −189 fragment (containing a CRE element) was cloned into pCD39/52 to make the deletion construct pCD39/ΔCRE.

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: .. It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. The sequences of inserted mCherry genes in the pENTR4 plasmids were read using sanger sequencing and confirmed to be correct sequences.

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Positive clones were cultured, and plasmid DNA isolated and inserts sequenced using the T7 primer as described above.

Amplification:

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1. .. Accessible or control regions located upstream of the genes PF3D7_0719000, PF3D7_1200700 and PF3D7_1222700 or the accessible region upstream of PF3D7_1372200 were amplified and inserted upstream of the kahrp minimal promoter using their respective primers listed in and BglII/Not I digestion and ligation by the T4 ligase.

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B). .. The PCR reaction was assembled in a total volume of 50 μl by adding 10 μl DNA, 2 μl Nextflex primer, 25 μl Kapa 2 × master mix (Kapa #KK2612), and 13 μl of H2 O. PCR was performed for 4 cycles using amplification conditions as mentioned in the Kapa protocol followed by purification using the Qiagen mini elute reaction clean up kit.

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: The mCherry gene, having restriction enzyme sites of 5′-EcoRI and 3′-NotI, was amplified by PCR with templated pcDNA3.1 –Peredox –mCherry . .. It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA).

Synthesized:

Article Title: BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease
Article Snippet: The PCR product was digested with EcoRI and BamHI and ligated into pEGFP-N3 using T4 ligase (Promega). .. For NLS-Tβ4-myc/pcDNA3: NLS-Tβ4-myc was synthesized by Eurofins MWG Operon and inserted into pcDNA3.

Article Title: Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus
Article Snippet: Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA). .. The ZnT8 optimized nucleotide sequence was synthesized by GenScript (GenScript Corporation, Piscataway, NJ, USA; http://www.GenScript.com ) including Kpn I and Xba I sites at the 3′ and 5′ ends, respectively.

TA Cloning:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: A blunt end cutting enzyme to Taq-generate T overhangs for TA cloning of PCR products or any other means to insert a fragment of choice are of course feasible as well. .. We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ).

Construct:

Article Title: Enzyme-guided DNA Sewing Architecture
Article Snippet: .. To construct T-DNA, sequences were formed via a complementary hybridization of each base, followed by T4 ligase (Promega, Madison, WI). ..

Article Title: BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease
Article Snippet: Paragraph title: DNA constructs and plasmids ... The PCR product was digested with EcoRI and BamHI and ligated into pEGFP-N3 using T4 ligase (Promega).

Article Title: Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus
Article Snippet: Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA). .. The synthesized construct (682 bp) was obtained from GenScript in plasmid pUC57 and maintained in E. coli strain JM109 (Promega, Madison, WI, USA).

Article Title: cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression *
Article Snippet: .. PCR was used to amplify mouse Cd39 promoter-luciferase reporter constructs representing 5′-deleted Cd39 upstream sequences, and these PCR products were subcloned into the pGL3-basic vector (Promega) using a T4 ligase and Quick Ligasing buffer (Promega). ..

Electrophoresis:

Article Title: Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *
Article Snippet: Unless otherwise stated, reactions (10 μl) contained 10 n m (molecules) DNA substrate in 20 m m HEPES-NaOH, pH 7.5, 100 μg/ml bovine serum albumin, 10% glycerol, 5 m m MgCl2 , 4 m m ATP, 4 μ m [α-32 P]dCTP (∼60 Ci/mmol), and the following proteins as indicated: UL2 (200 n m ), E. coli UDG (1 unit), APE (1.25 units), UL30 (100 n m ), UL42 (100 n m ), T4 ligase (Promega), ligase I (50 n m ), ligase IIIα-XRCC1 (50 n m ), Pol β (100 n m ), Pol δ (400 n m ), or exonuclease-deficient Klenow Pol (1 unit). .. After 40 min at 37 °C, the reactions were combined with an equal volume of stop buffer (95% formamide, 20 m m EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol), heated for 3 min at 75 °C, and chilled on ice before electrophoresis through 15% polyacrylamide, 8 m urea gels in GTG buffer (89 m m Tris, pH 9.0, 28.5 m m taurine, and 0.5 m m EDTA).

Incubation:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: .. We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ). .. We transformed 2 μl of the resulting solution into DH5α chemically competent E. coli (Invitrogen) and plated on two plates either containing kanamycin or ampicillin.

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: The genomic DNA was partially digested with Sau3A for 1 h at 37°C to obtain DNA fragments of around 20 to 30 kbp in size and then ligated overnight at 4°C to the pYUB412-derived 3,789-bp and 4,773-bp fragments using T4 ligase (Promega). .. The ligation reaction mixture (5 µl) was mixed with 25 µl of MaxPlax Lambda Packaging Extract (Epicentre, Madison, WI) at room temperature (RT) for 1 h. Another 2 µl of ligation reaction mixture was added to the packaging reaction, and the mixture was incubated for an additional hour at RT.

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: The reaction was incubated at 37 °C for 30 min followed by purification using the Qiagen mini elute reaction clean up kit. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: .. The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Recombinant pET30a(+) vectors containing inserts derived from the five pheromone binding proteins (Ofur PBP1-Ofur PBP5) were used to transform BL21(DE3) Escherichia coli cells by electroporation, and cells allowed to recover in SOC medium for 1 h at 37 °C.

Expressing:

Article Title: Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus
Article Snippet: Paragraph title: Expression vector ... Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA).

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: Paragraph title: Prokaryotic expression and purification ... The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C.

Modification:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: To test our method we accordingly modified the plasmid pGEM-T easy and included a BglII site to be able to insert a DNA fragment between these elements. .. We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ).

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: M. tuberculosis H37Rv genomic DNA was prepared from a 20-ml culture (OD600 , ~3) grown in Middlebrook 7H9 supplemented with 10% OADC, 0.2% glycerol, and 0.05% Tween 80 as previously described , with the following modification: the genomic DNA, once precipitated in isopropanol, was spooled and immersed in ethanol and was then air-dried instead of being spun down. .. The genomic DNA was partially digested with Sau3A for 1 h at 37°C to obtain DNA fragments of around 20 to 30 kbp in size and then ligated overnight at 4°C to the pYUB412-derived 3,789-bp and 4,773-bp fragments using T4 ligase (Promega).

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: To generate the specific att P-plasmids, the pDC2 plasmid ( ) was modified on several points. (All primers used for cloning, integration checking and RT-qPCR are listed in ). .. The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1.

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. Subsequently, a pAd /CMV /V5-DEST plasmid containing the mCherry gene was digested with Pac I restriction enzymes (New England Biolabs, Ipswich, MA, USA), and the resulting liner plasmids were transfected using Lipofectamine LTX Reagent (Thermo Fisher Scientific) into HEK 293A cells cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific), containing 10% Fetal Clone III (GE Healthcare, Chicago, IL, USA) and antibiotics (Nacalai tesque, Kyoto, Japan) to synthesize and amplify rAdV vectors containing the mCherry gene.

Transformation Assay:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ). .. We transformed 2 μl of the resulting solution into DH5α chemically competent E. coli (Invitrogen) and plated on two plates either containing kanamycin or ampicillin.

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Transformed cells were selected by spread plating on LB agar plates containing 25 μg ml−1 kanamycin sulfates as described by the manufacturer.

Derivative Assay:

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Recombinant pET30a(+) vectors containing inserts derived from the five pheromone binding proteins (Ofur PBP1-Ofur PBP5) were used to transform BL21(DE3) Escherichia coli cells by electroporation, and cells allowed to recover in SOC medium for 1 h at 37 °C.

Hybridization:

Article Title: Enzyme-guided DNA Sewing Architecture
Article Snippet: .. To construct T-DNA, sequences were formed via a complementary hybridization of each base, followed by T4 ligase (Promega, Madison, WI). ..

Electroporation:

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Recombinant pET30a(+) vectors containing inserts derived from the five pheromone binding proteins (Ofur PBP1-Ofur PBP5) were used to transform BL21(DE3) Escherichia coli cells by electroporation, and cells allowed to recover in SOC medium for 1 h at 37 °C.

Transfection:

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. Subsequently, a pAd /CMV /V5-DEST plasmid containing the mCherry gene was digested with Pac I restriction enzymes (New England Biolabs, Ipswich, MA, USA), and the resulting liner plasmids were transfected using Lipofectamine LTX Reagent (Thermo Fisher Scientific) into HEK 293A cells cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific), containing 10% Fetal Clone III (GE Healthcare, Chicago, IL, USA) and antibiotics (Nacalai tesque, Kyoto, Japan) to synthesize and amplify rAdV vectors containing the mCherry gene.

Inverse PCR:

Article Title: A long noncoding RNA promotes cellulase expression in Trichoderma reesei
Article Snippet: Paragraph title: Inverse PCR ... After heat inactivation, 2 µl of T4 Ligase (Promega, 1–3 U/µl) and the corresponding buffer were added and ligation was performed at 18 °C for 90 min.

Ligation:

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: The genomic DNA was partially digested with Sau3A for 1 h at 37°C to obtain DNA fragments of around 20 to 30 kbp in size and then ligated overnight at 4°C to the pYUB412-derived 3,789-bp and 4,773-bp fragments using T4 ligase (Promega). .. The ligation reaction mixture (5 µl) was mixed with 25 µl of MaxPlax Lambda Packaging Extract (Epicentre, Madison, WI) at room temperature (RT) for 1 h. Another 2 µl of ligation reaction mixture was added to the packaging reaction, and the mixture was incubated for an additional hour at RT.

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: .. The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1. .. The snf7-gfp element was replaced by the gfp-luc sequence from the MV163 plasmid ( ) using the primers ‘GFPLuc-F’ and ‘GFPLuc-R’, Avr II/Xho I digestion and ligation by the T4 ligase resulting in plasmid pOM2.

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: To prepare the DNA fragments for adaptor ligation an adenosine base was added to the 3′ ends of the repaired DNA by addition of 5 μl Klenow buffer, 10 μl dATP and 1 ul Klenow exo– (NEB # M0212L). .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: A long noncoding RNA promotes cellulase expression in Trichoderma reesei
Article Snippet: .. After heat inactivation, 2 µl of T4 Ligase (Promega, 1–3 U/µl) and the corresponding buffer were added and ligation was performed at 18 °C for 90 min. .. Subsequently, the ligation was stopped by heat inactivation and 1 µl was applied as template in a 25 µl inverse PCR reaction, initially using the primers amdS inv for and amdS inv rev.

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: .. It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. The sequences of inserted mCherry genes in the pENTR4 plasmids were read using sanger sequencing and confirmed to be correct sequences.

Cell Culture:

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. Subsequently, a pAd /CMV /V5-DEST plasmid containing the mCherry gene was digested with Pac I restriction enzymes (New England Biolabs, Ipswich, MA, USA), and the resulting liner plasmids were transfected using Lipofectamine LTX Reagent (Thermo Fisher Scientific) into HEK 293A cells cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific), containing 10% Fetal Clone III (GE Healthcare, Chicago, IL, USA) and antibiotics (Nacalai tesque, Kyoto, Japan) to synthesize and amplify rAdV vectors containing the mCherry gene.

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Positive clones were cultured, and plasmid DNA isolated and inserts sequenced using the T7 primer as described above.

Generated:

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: Plasmid DNA Cloning To examine the regulatory potential of the identified accessible several parasite lines were generated: four parasite lines with an integrated plasmid containing an accessible region detected by ATAC-seq upstream of a minimal kahrp promoter and a gfp-luc reporter gene ( A and A), two parasite lines with a not-accessible, control region instead of the accessible region (a third line did not show successful integration) and one parasite with an integrated plasmid containing the minimal promoter followed by the reporter gene. .. The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1.

Polymerase Chain Reaction:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: A blunt end cutting enzyme to Taq-generate T overhangs for TA cloning of PCR products or any other means to insert a fragment of choice are of course feasible as well. .. We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ).

Article Title: BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease
Article Snippet: .. The PCR product was digested with EcoRI and BamHI and ligated into pEGFP-N3 using T4 ligase (Promega). .. For NLS-Tβ4-myc/pcDNA3: NLS-Tβ4-myc was synthesized by Eurofins MWG Operon and inserted into pcDNA3.

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: End repair was performed using 40 μl of ChIP DNA, 5 μl T4 DNA ligase buffer with 10 mM ATP, 2 μl dNTP mix (10 mM each), 1 μl of T4 DNA polymerase (NEB # M0203L), 1 μl diluted Klenow DNA polymerase (1:5, NEB # M0210L), and T4 PNK (NEB # M0201L) in a total volume of 50 μl and incubation at 20 °C for 30 min, followed by purification using the QIAquick PCR purification kit and elution in 34 μl of EB. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression *
Article Snippet: .. PCR was used to amplify mouse Cd39 promoter-luciferase reporter constructs representing 5′-deleted Cd39 upstream sequences, and these PCR products were subcloned into the pGL3-basic vector (Promega) using a T4 ligase and Quick Ligasing buffer (Promega). ..

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: The mCherry gene, having restriction enzyme sites of 5′-EcoRI and 3′-NotI, was amplified by PCR with templated pcDNA3.1 –Peredox –mCherry . .. It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA).

Recombinant:

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: Paragraph title: 2.1. Cloning of Recombinant Adenoviral Vectors Containing the mCherry Gene ... It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA).

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Recombinant pET30a(+) vectors containing inserts derived from the five pheromone binding proteins (Ofur PBP1-Ofur PBP5) were used to transform BL21(DE3) Escherichia coli cells by electroporation, and cells allowed to recover in SOC medium for 1 h at 37 °C.

Nucleic Acid Purification:

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: Prokaryotic expression and purification The pGEM plasmid inserts were excised using Bam HI and Hind III double digestion, excised from a 1% agarose gel and fragments purified using Nucleic Acid Purification Kit (Axygen, USA). .. The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C.

ChIP-sequencing:

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B). .. The reaction was incubated at room temperature for 15 min. DNA was purified using the Qiagen mini elute reaction clean up kit, eluted in 10 μl of EB, and followed by PCR.

Mutagenesis:

Article Title: cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression *
Article Snippet: Paragraph title: Construction of Mouse Cd39 Promoter-Luciferase Reporter Plasmids and Site-directed Mutagenesis ... PCR was used to amplify mouse Cd39 promoter-luciferase reporter constructs representing 5′-deleted Cd39 upstream sequences, and these PCR products were subcloned into the pGL3-basic vector (Promega) using a T4 ligase and Quick Ligasing buffer (Promega).

Isolation:

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Positive clones were cultured, and plasmid DNA isolated and inserts sequenced using the T7 primer as described above.

Subcloning:

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: Paragraph title: M. tuberculosis cosmid library construction and subcloning analysis of TNF-downregulating hits. ... The genomic DNA was partially digested with Sau3A for 1 h at 37°C to obtain DNA fragments of around 20 to 30 kbp in size and then ligated overnight at 4°C to the pYUB412-derived 3,789-bp and 4,773-bp fragments using T4 ligase (Promega).

Purification:

Article Title: Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins
Article Snippet: In short, the ECs were assembled using purified yeast Pol II and DNA/RNA oligonucleotides. .. ECs and nucleosomal templates (or corresponding DNA fragments) were ligated by T4 ligase (Promega).

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: The reaction was incubated at 37 °C for 30 min followed by purification using the Qiagen mini elute reaction clean up kit. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: Paragraph title: Prokaryotic expression and purification ... The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C.

Sequencing:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: Both of the two identical key elements comprise a recognition sequence for a type IIs restriction endonuclease – Esp3I in our case – and a restriction site (Figure ). .. We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ).

Article Title: Enzyme-guided DNA Sewing Architecture
Article Snippet: Synthesis of model DNA sewing materials To synthesize a Y-DNA block, three different oligonucleotides were designed and manufactured. demonstrates the T-DNA and Y-DNA sequence information. .. To construct T-DNA, sequences were formed via a complementary hybridization of each base, followed by T4 ligase (Promega, Madison, WI).

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1. .. The snf7-gfp element was replaced by the gfp-luc sequence from the MV163 plasmid ( ) using the primers ‘GFPLuc-F’ and ‘GFPLuc-R’, Avr II/Xho I digestion and ligation by the T4 ligase resulting in plasmid pOM2.

Article Title: BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease
Article Snippet: Tβ4-EGFP-N3: using a full-length cDNA of Tβ4 in pcDNA3.1 as template DNA, PCR was performed using primers that added a Kozak sequence and EcoRI site to the 5′ end and a flexible glycine-serine linker and BamHI site to the 3′ end of the Tβ4 open-reading frame (ORF). .. The PCR product was digested with EcoRI and BamHI and ligated into pEGFP-N3 using T4 ligase (Promega).

Article Title: Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus
Article Snippet: Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA). .. The coding sequence of dimeric C-terminal domain of human ZnT8 consisting of amino acids 268–369 [Arg325] and amino acids 268–369 [Trp325] were optimized for prokaryotic expression.

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. The sequences of inserted mCherry genes in the pENTR4 plasmids were read using sanger sequencing and confirmed to be correct sequences.

Quantitative RT-PCR:

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: To generate the specific att P-plasmids, the pDC2 plasmid ( ) was modified on several points. (All primers used for cloning, integration checking and RT-qPCR are listed in ). .. The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1.

Chromatin Immunoprecipitation:

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: End repair was performed using 40 μl of ChIP DNA, 5 μl T4 DNA ligase buffer with 10 mM ATP, 2 μl dNTP mix (10 mM each), 1 μl of T4 DNA polymerase (NEB # M0203L), 1 μl diluted Klenow DNA polymerase (1:5, NEB # M0210L), and T4 PNK (NEB # M0201L) in a total volume of 50 μl and incubation at 20 °C for 30 min, followed by purification using the QIAquick PCR purification kit and elution in 34 μl of EB. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Plasmid Preparation:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: .. We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ). .. We transformed 2 μl of the resulting solution into DH5α chemically competent E. coli (Invitrogen) and plated on two plates either containing kanamycin or ampicillin.

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: The plasmid pYUB412 was linearized with XbaI, dephosphorylated with shrimp alkaline phosphatase (Promega), and then digested with BclI to obtain 3,789-bp and 4,773-bp fragments. .. The genomic DNA was partially digested with Sau3A for 1 h at 37°C to obtain DNA fragments of around 20 to 30 kbp in size and then ligated overnight at 4°C to the pYUB412-derived 3,789-bp and 4,773-bp fragments using T4 ligase (Promega).

Article Title: Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development
Article Snippet: .. The orientation of the 5′cam-snf7-gfp-3′hsp86 cassette was reversed using the primers ‘5′Pfcam-F’ and ‘3′hsp86-R’, PstI/Apa I digestion and ligation by the T4 ligase (Promega, #M1804) resulting in plasmid pOM1. .. The snf7-gfp element was replaced by the gfp-luc sequence from the MV163 plasmid ( ) using the primers ‘GFPLuc-F’ and ‘GFPLuc-R’, Avr II/Xho I digestion and ligation by the T4 ligase resulting in plasmid pOM2.

Article Title: Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus
Article Snippet: Paragraph title: Expression vector ... Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA).

Article Title: cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression *
Article Snippet: .. PCR was used to amplify mouse Cd39 promoter-luciferase reporter constructs representing 5′-deleted Cd39 upstream sequences, and these PCR products were subcloned into the pGL3-basic vector (Promega) using a T4 ligase and Quick Ligasing buffer (Promega). ..

Article Title: Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Article Snippet: .. It was then cloned into a pENTR4 plasmid between EcoRI and NotI sites by restriction enzyme digestion, followed by ligation with T4 ligase (Promega, Madison, WI, USA). .. The sequences of inserted mCherry genes in the pENTR4 plasmids were read using sanger sequencing and confirmed to be correct sequences.

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: .. The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Recombinant pET30a(+) vectors containing inserts derived from the five pheromone binding proteins (Ofur PBP1-Ofur PBP5) were used to transform BL21(DE3) Escherichia coli cells by electroporation, and cells allowed to recover in SOC medium for 1 h at 37 °C.

Binding Assay:

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C. .. Recombinant pET30a(+) vectors containing inserts derived from the five pheromone binding proteins (Ofur PBP1-Ofur PBP5) were used to transform BL21(DE3) Escherichia coli cells by electroporation, and cells allowed to recover in SOC medium for 1 h at 37 °C.

Agarose Gel Electrophoresis:

Article Title: Binding affinity of five PBPs to Ostrinia sex pheromones
Article Snippet: Prokaryotic expression and purification The pGEM plasmid inserts were excised using Bam HI and Hind III double digestion, excised from a 1% agarose gel and fragments purified using Nucleic Acid Purification Kit (Axygen, USA). .. The Bam HI and Hind III digestion products for Ofur PBP1 to Ofur PBP5 were then ligated individually into Bam HI and Hind III cut and dephosphorylated pET30a(+) vector by incubation with T4 Ligase (Promega) for 4 h at 16 °C.

In Vitro:

Article Title: Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins
Article Snippet: Paragraph title: In vitro transcription assay ... ECs and nucleosomal templates (or corresponding DNA fragments) were ligated by T4 ligase (Promega).

Next-Generation Sequencing:

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: Paragraph title: Illumina high throughput sequencing ... Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Concentration Assay:

Article Title: Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins
Article Snippet: ECs and nucleosomal templates (or corresponding DNA fragments) were ligated by T4 ligase (Promega). .. ECs were washed from the unincorporated NTPs and eluted, and transcription was resumed in the presence of unlabeled NTPs, hFACT (final concentration, 0.1 μM), CBL0137 (1 μM) or CBL0100 (5 μM), and mononucleosomes (~0.18 μM) in the TB containing 150 mM KCl for 10 min.

Article Title: A long noncoding RNA promotes cellulase expression in Trichoderma reesei
Article Snippet: 20 µg of chromosomal DNA were digested with either Acc 65I or Not I (Thermo Scientific) at a final concentration of 1 U/µl in a total reaction volume of 20 µl according to the manufacturer’s instructions. .. After heat inactivation, 2 µl of T4 Ligase (Promega, 1–3 U/µl) and the corresponding buffer were added and ligation was performed at 18 °C for 90 min.

Marker:

Article Title: A long noncoding RNA promotes cellulase expression in Trichoderma reesei
Article Snippet: Inverse PCR For the identification of the locus that was targeted by integration of the amdS marker in QM9414_Dhax1 strains, an inverse PCR was performed as follows. .. After heat inactivation, 2 µl of T4 Ligase (Promega, 1–3 U/µl) and the corresponding buffer were added and ligation was performed at 18 °C for 90 min.

Blocking Assay:

Article Title: Enzyme-guided DNA Sewing Architecture
Article Snippet: Synthesis of model DNA sewing materials To synthesize a Y-DNA block, three different oligonucleotides were designed and manufactured. demonstrates the T-DNA and Y-DNA sequence information. .. To construct T-DNA, sequences were formed via a complementary hybridization of each base, followed by T4 ligase (Promega, Madison, WI).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Promega t4 rna ligase
    Enzymatic determination of the new 3′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) <t>T4</t> RNA ligase treatment of gel-purified HCV RNA 1–130 (left panel) and CSFV RNA 1–218 (right panel) cleavage product band B2. B2 RNAs [4000 dpm (10 5 dpm/µg)] were incubated with T4 RNA ligase and [5′- 32 P]pCp. Lane 1: control reaction with B2 RNA incubated in SAP phosphatase buffer, then in ligase buffer and [5′- 32 P]pCp in the absence of any enzyme; Lane 2: control reaction of B2 RNA treated the same as in lane 1 but incubated with the phosphatase; Lane 3: B2 RNA incubated with T4 RNA ligase without previous dephosphorylation; Lane 4: complete reaction of B2 RNA incubated with the ligase after being treated with the phosphatase. ( b ) Addition of [ 32 P]-labeled poly (A) or poly (U) to bands B2 of HCV (left panel) and CSFV (right panel) with E. coli poly (A) polymerase or Schizosaccharomyces pombe poly (U) polymerase.A total of 4000 dpm RNA (10 5 dpm/µg) was used for both viral RNAs. A total of 20 µCi of the labeled nucleotide (ATP or UTP) was distributed for the four reactions. Lanes 1 and 2: B2 RNA incubated with the poly (A) polymerase after being treated or not with shrimp alkaline phosphatase, respectively. Lanes 3 and 4: control reactions of B2 RNA treated or not with the phosphatase but without incubation with the polymerase. Lanes 1′ 2′ 3′ and 4′ same as above, but using poly (U) polymerase. MW is a molecular weight marker.
    T4 Rna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/Promega
    Average 94 stars, based on 48 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-01
    94/100 stars
      Buy from Supplier

    96
    Promega t4 ligase
    Time course of BER. 35-μl reactions were performed as described under “Experimental Procedures” with UL2, APE, and UL30 in the presence of either <t>T4</t> ligase (0.001 units/μl) ( A ), ligase I ( B ), or ligase IIIα-XRCC1 ( C ). 5-μl aliquots were removed at the times indicated. Activity is expressed as the fraction of maximum for the nicked ( N ) (●) and ligation ( L ) (○) products. The insets in each panel show the relevant gel images.
    T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/Promega
    Average 96 stars, based on 102 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-01
    96/100 stars
      Buy from Supplier

    98
    Promega t4 dna ligase
    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Promega
    Average 98 stars, based on 397 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-01
    98/100 stars
      Buy from Supplier

    Image Search Results


    Enzymatic determination of the new 3′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) T4 RNA ligase treatment of gel-purified HCV RNA 1–130 (left panel) and CSFV RNA 1–218 (right panel) cleavage product band B2. B2 RNAs [4000 dpm (10 5 dpm/µg)] were incubated with T4 RNA ligase and [5′- 32 P]pCp. Lane 1: control reaction with B2 RNA incubated in SAP phosphatase buffer, then in ligase buffer and [5′- 32 P]pCp in the absence of any enzyme; Lane 2: control reaction of B2 RNA treated the same as in lane 1 but incubated with the phosphatase; Lane 3: B2 RNA incubated with T4 RNA ligase without previous dephosphorylation; Lane 4: complete reaction of B2 RNA incubated with the ligase after being treated with the phosphatase. ( b ) Addition of [ 32 P]-labeled poly (A) or poly (U) to bands B2 of HCV (left panel) and CSFV (right panel) with E. coli poly (A) polymerase or Schizosaccharomyces pombe poly (U) polymerase.A total of 4000 dpm RNA (10 5 dpm/µg) was used for both viral RNAs. A total of 20 µCi of the labeled nucleotide (ATP or UTP) was distributed for the four reactions. Lanes 1 and 2: B2 RNA incubated with the poly (A) polymerase after being treated or not with shrimp alkaline phosphatase, respectively. Lanes 3 and 4: control reactions of B2 RNA treated or not with the phosphatase but without incubation with the polymerase. Lanes 1′ 2′ 3′ and 4′ same as above, but using poly (U) polymerase. MW is a molecular weight marker.

    Journal: Nucleic Acids Research

    Article Title: RNA self-cleavage activated by ultraviolet light-induced oxidation

    doi: 10.1093/nar/gkr822

    Figure Lengend Snippet: Enzymatic determination of the new 3′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) T4 RNA ligase treatment of gel-purified HCV RNA 1–130 (left panel) and CSFV RNA 1–218 (right panel) cleavage product band B2. B2 RNAs [4000 dpm (10 5 dpm/µg)] were incubated with T4 RNA ligase and [5′- 32 P]pCp. Lane 1: control reaction with B2 RNA incubated in SAP phosphatase buffer, then in ligase buffer and [5′- 32 P]pCp in the absence of any enzyme; Lane 2: control reaction of B2 RNA treated the same as in lane 1 but incubated with the phosphatase; Lane 3: B2 RNA incubated with T4 RNA ligase without previous dephosphorylation; Lane 4: complete reaction of B2 RNA incubated with the ligase after being treated with the phosphatase. ( b ) Addition of [ 32 P]-labeled poly (A) or poly (U) to bands B2 of HCV (left panel) and CSFV (right panel) with E. coli poly (A) polymerase or Schizosaccharomyces pombe poly (U) polymerase.A total of 4000 dpm RNA (10 5 dpm/µg) was used for both viral RNAs. A total of 20 µCi of the labeled nucleotide (ATP or UTP) was distributed for the four reactions. Lanes 1 and 2: B2 RNA incubated with the poly (A) polymerase after being treated or not with shrimp alkaline phosphatase, respectively. Lanes 3 and 4: control reactions of B2 RNA treated or not with the phosphatase but without incubation with the polymerase. Lanes 1′ 2′ 3′ and 4′ same as above, but using poly (U) polymerase. MW is a molecular weight marker.

    Article Snippet: RNA ligase was used to treat cleavage product bands, either previously phosphatase treated or not, as follows: RNAs were incubated at 4°C for 4 days in a buffer containing 10 mM MgCl2 , 50 mM HEPES, pH 8.3, 5 mM DTT, 0.12 mM ATP, 4 U of T4 RNA ligase (Promega) and [5′-32 P]pCp (Perkin-Elmer) ( , ).

    Techniques: Produced, Purification, Incubation, De-Phosphorylation Assay, Labeling, Molecular Weight, Marker

    Characterization of UV-C cleavage of viral RNAs by fingerprinting and electrophoretic methods. ( a ) The RNA fingerprints of internally [α- 32 P] HCV (panels 1–3) and CSFV RNA (panels 4–6). Labeled SM, band B1 and band B2 were exhaustively digested with RNase T1 and the products subjected to 2D separation. ( 1 ) RNA fingerprint of HCV 1–249. A total of 500 000 dpm of HCV SM was fingerprinted. Spot 1: the HCV oligonucleotide 5′ 78 UCUAG 82 3′ within which cleavage takes place; Spot 2: this has the characteristic mobility of the 5′-terminal nucleotide 5′pppGp3′. ( 2 ) RNA fingerprint of HCV B1. A total of 300 000 dpm of RNA was fingerprinted as above. Spot 1 has disappeared, while the absence of the 5′-end (spot 2) shows that HCV B1 contains the 3′-portion of SM. ( 3 ) Fingerprint of HCV B2 (100 000 dpm). ‘1’ indicates the loss of spot 1, while the presence of the HCV 5′-end (‘2’) shows that HCV B2 contains the 5′-portion of SM. ( 4–6 ): RNA fingerprints of CSFV 1-218. SM (500 000 dpm), B1 (300 000 dpm) and B2 (100 000 dpm, transcribed with all four [α- 32 P]-labeled rNTPs. ‘1’: the CSFV oligonucleotide 5′ 38 AUACACUAAAUUUCG 52 3′, which is present in SM but absent from B1 and B2; ‘X’ (a new CSFV B1 oligonucleotide) and ‘Y’ (the other new CSFV oligonucleotide) arise from cleavage within spot 1 (see text) . ‘2’: the 5′-end of CSFV, present in SM and B2, but not B1. Numbering according to Wang et al. ( 62 ) for HCV genotype 1b and Gene Bank J04358 for CSFV Alfort Isolate. The sequence of the spot numbered as 1 was identified by secondary RNase analysis and high voltage electrophoresis on DEAE and Whatmann paper by Hugh D. Robertson (data not available), as well as by superposition with previously resolved HCV fingerprints using secondary analysis and on the basis of the rules for RNA oligonucleotide mobility during 2D TLC. Briefly, these rules are: the larger the oligonucleotide, the slower the migration of the corresponding spot to the bottom. As far as composition is concerned, Us displace the spot to the left, Cs to the right, and As cause a slight delay, thus meaning that several As in the same oligo may cause it to behave as an oligo containing one or even two additional bases ( 37 ). As far as sequence is concerned, as HCV RNA was transcribed in the presence of [α- 32 P]GTP here, those T1 oligonucleotides in the original sequence that are followed by a (pG) carry a double label. In the case of HCV RNA several RNase T1 oligonucelotides are indicated as mobility reference: a: UCCUUUCUUGp(G); b: UCUUCAGp(C) 61:68; c: CUCAAUGp(G) 211–217; d: AUUUGp(G) 225–229. Spot 1 locates in the border of the triangle that can be formed by spots i, f and g. In CSFV, spot 1 is the slow migrating spot, and thus corresponded to the largest RNase T1 oligonucleotide. In both HCV and CSFV, band B2 contains the original 5′-terminal nucleotide, pppGp, of the substrate RNA transcript (indicated by ‘2’). The disappearance of spot 1 from both product band fingerprints (see Figure 1 a, images 2, 3 and images 5, 6) suggests that self-cleavage occurs within this oligonucleotide and that this event is specific. Moreover, in the case of CSFV RNA two smaller oligomers (X and Y) that represent the fragment products of spot 1 are observed within the fingerprints of both product bands (B1 and B2) for CSFV. Indicated at the bottom is the sequence surrounding RNase T1 cleavage sites. ( b ) Electrophoresis analysis: Autoradiogram showing a parallel run of HCV RNA 1–130 and CSFV RNA 1–218 UV-cleavage reaction, with RNase T1 treated samples and control reactions for transcripts labeled at either the 5′-extreme with [γ- 32 P]GTP during transcription or the 3′-extreme with [5′- 32 P]pCp and T4 RNA ligase. HCV (lanes 1–6) and CSFV (lanes 1–9). HCV: Lanes 1 and 1′: RNAs incubated in standard buffer; lanes 2 and 2′: RNAs treated with RNase T1 under denaturing conditions; lanes 3 and 3′: RNA irradiated with UV-C light for 180 s. CSFV: Lanes 1 and 1′: RNAs incubated under standard conditions; lanes 2, 3 and 4′: RNA treated with RNase T1; lanes 4 and 3′: RNA irradiated with UV-C light: Lanes 5 and 2′ RNAs partially degraded with alkali. ‘G’ positions of a relevant size are indicated on either side of the gels. Lines indicate SM, and products B1 and B2.

    Journal: Nucleic Acids Research

    Article Title: RNA self-cleavage activated by ultraviolet light-induced oxidation

    doi: 10.1093/nar/gkr822

    Figure Lengend Snippet: Characterization of UV-C cleavage of viral RNAs by fingerprinting and electrophoretic methods. ( a ) The RNA fingerprints of internally [α- 32 P] HCV (panels 1–3) and CSFV RNA (panels 4–6). Labeled SM, band B1 and band B2 were exhaustively digested with RNase T1 and the products subjected to 2D separation. ( 1 ) RNA fingerprint of HCV 1–249. A total of 500 000 dpm of HCV SM was fingerprinted. Spot 1: the HCV oligonucleotide 5′ 78 UCUAG 82 3′ within which cleavage takes place; Spot 2: this has the characteristic mobility of the 5′-terminal nucleotide 5′pppGp3′. ( 2 ) RNA fingerprint of HCV B1. A total of 300 000 dpm of RNA was fingerprinted as above. Spot 1 has disappeared, while the absence of the 5′-end (spot 2) shows that HCV B1 contains the 3′-portion of SM. ( 3 ) Fingerprint of HCV B2 (100 000 dpm). ‘1’ indicates the loss of spot 1, while the presence of the HCV 5′-end (‘2’) shows that HCV B2 contains the 5′-portion of SM. ( 4–6 ): RNA fingerprints of CSFV 1-218. SM (500 000 dpm), B1 (300 000 dpm) and B2 (100 000 dpm, transcribed with all four [α- 32 P]-labeled rNTPs. ‘1’: the CSFV oligonucleotide 5′ 38 AUACACUAAAUUUCG 52 3′, which is present in SM but absent from B1 and B2; ‘X’ (a new CSFV B1 oligonucleotide) and ‘Y’ (the other new CSFV oligonucleotide) arise from cleavage within spot 1 (see text) . ‘2’: the 5′-end of CSFV, present in SM and B2, but not B1. Numbering according to Wang et al. ( 62 ) for HCV genotype 1b and Gene Bank J04358 for CSFV Alfort Isolate. The sequence of the spot numbered as 1 was identified by secondary RNase analysis and high voltage electrophoresis on DEAE and Whatmann paper by Hugh D. Robertson (data not available), as well as by superposition with previously resolved HCV fingerprints using secondary analysis and on the basis of the rules for RNA oligonucleotide mobility during 2D TLC. Briefly, these rules are: the larger the oligonucleotide, the slower the migration of the corresponding spot to the bottom. As far as composition is concerned, Us displace the spot to the left, Cs to the right, and As cause a slight delay, thus meaning that several As in the same oligo may cause it to behave as an oligo containing one or even two additional bases ( 37 ). As far as sequence is concerned, as HCV RNA was transcribed in the presence of [α- 32 P]GTP here, those T1 oligonucleotides in the original sequence that are followed by a (pG) carry a double label. In the case of HCV RNA several RNase T1 oligonucelotides are indicated as mobility reference: a: UCCUUUCUUGp(G); b: UCUUCAGp(C) 61:68; c: CUCAAUGp(G) 211–217; d: AUUUGp(G) 225–229. Spot 1 locates in the border of the triangle that can be formed by spots i, f and g. In CSFV, spot 1 is the slow migrating spot, and thus corresponded to the largest RNase T1 oligonucleotide. In both HCV and CSFV, band B2 contains the original 5′-terminal nucleotide, pppGp, of the substrate RNA transcript (indicated by ‘2’). The disappearance of spot 1 from both product band fingerprints (see Figure 1 a, images 2, 3 and images 5, 6) suggests that self-cleavage occurs within this oligonucleotide and that this event is specific. Moreover, in the case of CSFV RNA two smaller oligomers (X and Y) that represent the fragment products of spot 1 are observed within the fingerprints of both product bands (B1 and B2) for CSFV. Indicated at the bottom is the sequence surrounding RNase T1 cleavage sites. ( b ) Electrophoresis analysis: Autoradiogram showing a parallel run of HCV RNA 1–130 and CSFV RNA 1–218 UV-cleavage reaction, with RNase T1 treated samples and control reactions for transcripts labeled at either the 5′-extreme with [γ- 32 P]GTP during transcription or the 3′-extreme with [5′- 32 P]pCp and T4 RNA ligase. HCV (lanes 1–6) and CSFV (lanes 1–9). HCV: Lanes 1 and 1′: RNAs incubated in standard buffer; lanes 2 and 2′: RNAs treated with RNase T1 under denaturing conditions; lanes 3 and 3′: RNA irradiated with UV-C light for 180 s. CSFV: Lanes 1 and 1′: RNAs incubated under standard conditions; lanes 2, 3 and 4′: RNA treated with RNase T1; lanes 4 and 3′: RNA irradiated with UV-C light: Lanes 5 and 2′ RNAs partially degraded with alkali. ‘G’ positions of a relevant size are indicated on either side of the gels. Lines indicate SM, and products B1 and B2.

    Article Snippet: RNA ligase was used to treat cleavage product bands, either previously phosphatase treated or not, as follows: RNAs were incubated at 4°C for 4 days in a buffer containing 10 mM MgCl2 , 50 mM HEPES, pH 8.3, 5 mM DTT, 0.12 mM ATP, 4 U of T4 RNA ligase (Promega) and [5′-32 P]pCp (Perkin-Elmer) ( , ).

    Techniques: Labeling, Sequencing, Electrophoresis, Thin Layer Chromatography, Migration, Incubation, Irradiation

    Enzymatic determination of the new 5′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) Phosphatase-dependent 5′-terminal labeling of both HCV RNA 1–130 cleavage product (B1) (left panel) and CSFV RNA 1–218 cleavage product (B1) (right panel) by polynucleotide kinase. Aliquots (10 000 dpm) of product bands (10 5 dpm/µg) were treated with polynucleotide kinase and [γ- 32 P]ATP, after treatment with Artic Phosphatase (lane 2) or without phosphatase pre-treatment (lane 1). ( b ) Cyclization of HCV (left panel) and CSFV (right panel) RNA product bands B1 by T4 RNA ligase [with 10 000 dpm (10 5 dpm/µg) RNA]. Lanes 1: HCV and CSFV B1 bands incubated without T4 RNA ligase; lanes 2: complete reaction (cyclized RNA: upper band). ( c ) Phosphatase treatment of singly labeled CSFV. Calf alkaline phosphatase was used to treat CSFV band B1 aliquots (50 000 dpm of 107 dpm/µg), followed by high-voltage electrophoresis at pH 1.9 on Whatman DE81 DEAE paper ( 16 ). B1 RNA is at the bottom and free phosphate at the top. ‘U’, ‘A’, ‘C’ and ‘G’ indicate RNAs labeled with [α- 32 P]UTP, ATP, CTP or GTP, respectively, whereas ‘αGTP’ indicates a control containing 1000 dpm of pure [α- 32 P]GTP.

    Journal: Nucleic Acids Research

    Article Title: RNA self-cleavage activated by ultraviolet light-induced oxidation

    doi: 10.1093/nar/gkr822

    Figure Lengend Snippet: Enzymatic determination of the new 5′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) Phosphatase-dependent 5′-terminal labeling of both HCV RNA 1–130 cleavage product (B1) (left panel) and CSFV RNA 1–218 cleavage product (B1) (right panel) by polynucleotide kinase. Aliquots (10 000 dpm) of product bands (10 5 dpm/µg) were treated with polynucleotide kinase and [γ- 32 P]ATP, after treatment with Artic Phosphatase (lane 2) or without phosphatase pre-treatment (lane 1). ( b ) Cyclization of HCV (left panel) and CSFV (right panel) RNA product bands B1 by T4 RNA ligase [with 10 000 dpm (10 5 dpm/µg) RNA]. Lanes 1: HCV and CSFV B1 bands incubated without T4 RNA ligase; lanes 2: complete reaction (cyclized RNA: upper band). ( c ) Phosphatase treatment of singly labeled CSFV. Calf alkaline phosphatase was used to treat CSFV band B1 aliquots (50 000 dpm of 107 dpm/µg), followed by high-voltage electrophoresis at pH 1.9 on Whatman DE81 DEAE paper ( 16 ). B1 RNA is at the bottom and free phosphate at the top. ‘U’, ‘A’, ‘C’ and ‘G’ indicate RNAs labeled with [α- 32 P]UTP, ATP, CTP or GTP, respectively, whereas ‘αGTP’ indicates a control containing 1000 dpm of pure [α- 32 P]GTP.

    Article Snippet: RNA ligase was used to treat cleavage product bands, either previously phosphatase treated or not, as follows: RNAs were incubated at 4°C for 4 days in a buffer containing 10 mM MgCl2 , 50 mM HEPES, pH 8.3, 5 mM DTT, 0.12 mM ATP, 4 U of T4 RNA ligase (Promega) and [5′-32 P]pCp (Perkin-Elmer) ( , ).

    Techniques: Produced, Labeling, Incubation, Electrophoresis

    Time course of BER. 35-μl reactions were performed as described under “Experimental Procedures” with UL2, APE, and UL30 in the presence of either T4 ligase (0.001 units/μl) ( A ), ligase I ( B ), or ligase IIIα-XRCC1 ( C ). 5-μl aliquots were removed at the times indicated. Activity is expressed as the fraction of maximum for the nicked ( N ) (●) and ligation ( L ) (○) products. The insets in each panel show the relevant gel images.

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *

    doi: 10.1074/jbc.M109.010413

    Figure Lengend Snippet: Time course of BER. 35-μl reactions were performed as described under “Experimental Procedures” with UL2, APE, and UL30 in the presence of either T4 ligase (0.001 units/μl) ( A ), ligase I ( B ), or ligase IIIα-XRCC1 ( C ). 5-μl aliquots were removed at the times indicated. Activity is expressed as the fraction of maximum for the nicked ( N ) (●) and ligation ( L ) (○) products. The insets in each panel show the relevant gel images.

    Article Snippet: Unless otherwise stated, reactions (10 μl) contained 10 n m (molecules) DNA substrate in 20 m m HEPES-NaOH, pH 7.5, 100 μg/ml bovine serum albumin, 10% glycerol, 5 m m MgCl2 , 4 m m ATP, 4 μ m [α-32 P]dCTP (∼60 Ci/mmol), and the following proteins as indicated: UL2 (200 n m ), E. coli UDG (1 unit), APE (1.25 units), UL30 (100 n m ), UL42 (100 n m ), T4 ligase (Promega), ligase I (50 n m ), ligase IIIα-XRCC1 (50 n m ), Pol β (100 n m ), Pol δ (400 n m ), or exonuclease-deficient Klenow Pol (1 unit).

    Techniques: Activity Assay, Ligation

    Completion of BER; formation of ligation product. Reactions were performed as described under “Experimental Procedures” with the indicated proteins. Storage phosphorimage of reaction products obtained with UL2, APE, and UL30 ( lane 1 ) and identical reactions supplemented with T4 ligase (1.5 units) ( lane 2 ), ligase I ( lane 3 ), or ligase IIIα-XRCC1 ( lane 4 ) are shown. Lane 5 , DNA only; lane 6 , linear 100-mer. The positions of nicked product ( N ), ligated product ( L ), and of the 100-mer are as indicated. The values in italics below the lane numbers indicate the L/N ratios.

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *

    doi: 10.1074/jbc.M109.010413

    Figure Lengend Snippet: Completion of BER; formation of ligation product. Reactions were performed as described under “Experimental Procedures” with the indicated proteins. Storage phosphorimage of reaction products obtained with UL2, APE, and UL30 ( lane 1 ) and identical reactions supplemented with T4 ligase (1.5 units) ( lane 2 ), ligase I ( lane 3 ), or ligase IIIα-XRCC1 ( lane 4 ) are shown. Lane 5 , DNA only; lane 6 , linear 100-mer. The positions of nicked product ( N ), ligated product ( L ), and of the 100-mer are as indicated. The values in italics below the lane numbers indicate the L/N ratios.

    Article Snippet: Unless otherwise stated, reactions (10 μl) contained 10 n m (molecules) DNA substrate in 20 m m HEPES-NaOH, pH 7.5, 100 μg/ml bovine serum albumin, 10% glycerol, 5 m m MgCl2 , 4 m m ATP, 4 μ m [α-32 P]dCTP (∼60 Ci/mmol), and the following proteins as indicated: UL2 (200 n m ), E. coli UDG (1 unit), APE (1.25 units), UL30 (100 n m ), UL42 (100 n m ), T4 ligase (Promega), ligase I (50 n m ), ligase IIIα-XRCC1 (50 n m ), Pol β (100 n m ), Pol δ (400 n m ), or exonuclease-deficient Klenow Pol (1 unit).

    Techniques: Ligation

    Product formation is dependent on UL2, APE, UL30, and ligase. Storage phosphorimages of reactions performed as described under “Experimental Procedures” with the indicated components. Lane 1 , UL2; lane 2 , APE; lane 3 ; UL30; lane 4 , T4 ligase (1.5 units); lane 5 , ligase I; lane 6 , ligase IIIα-XRCC1; lane 7 , UL2 and APE; lane 8 , APE and UL30; lane 9 , UL2 and UL30; lane 10 , UL2, APE and UL30; lane 11 , UL2, APE, UL30 and T4 ligase (1.5 units); lane 12 , UL2, APE, UL30, and ligase I; lane 13 , UL2, APE, UL30, and ligase IIIα-XRCC1; lane 14 , DNA only. The positions of nicked ( N ) and ligated ( L ) products are as indicated.

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *

    doi: 10.1074/jbc.M109.010413

    Figure Lengend Snippet: Product formation is dependent on UL2, APE, UL30, and ligase. Storage phosphorimages of reactions performed as described under “Experimental Procedures” with the indicated components. Lane 1 , UL2; lane 2 , APE; lane 3 ; UL30; lane 4 , T4 ligase (1.5 units); lane 5 , ligase I; lane 6 , ligase IIIα-XRCC1; lane 7 , UL2 and APE; lane 8 , APE and UL30; lane 9 , UL2 and UL30; lane 10 , UL2, APE and UL30; lane 11 , UL2, APE, UL30 and T4 ligase (1.5 units); lane 12 , UL2, APE, UL30, and ligase I; lane 13 , UL2, APE, UL30, and ligase IIIα-XRCC1; lane 14 , DNA only. The positions of nicked ( N ) and ligated ( L ) products are as indicated.

    Article Snippet: Unless otherwise stated, reactions (10 μl) contained 10 n m (molecules) DNA substrate in 20 m m HEPES-NaOH, pH 7.5, 100 μg/ml bovine serum albumin, 10% glycerol, 5 m m MgCl2 , 4 m m ATP, 4 μ m [α-32 P]dCTP (∼60 Ci/mmol), and the following proteins as indicated: UL2 (200 n m ), E. coli UDG (1 unit), APE (1.25 units), UL30 (100 n m ), UL42 (100 n m ), T4 ligase (Promega), ligase I (50 n m ), ligase IIIα-XRCC1 (50 n m ), Pol β (100 n m ), Pol δ (400 n m ), or exonuclease-deficient Klenow Pol (1 unit).

    Techniques:

    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Article Snippet: The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

    Techniques: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

    DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Article Snippet: The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

    Techniques: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography