Structured Review

Promega t4 ligase
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 <t>T4</t> 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.
T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/t4 ligase/product/Promega
Average 95 stars, based on 165 article reviews
Price from $9.99 to $1999.99
t4 ligase - by Bioz Stars, 2020-05
95/100 stars

Images

1) 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

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 "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

4) 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

5) 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:

6) 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

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.

other:

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Article Snippet: Enzyme amounts: 7.5 units Esp3I, 10 units NheI, 3 weiss units T4 ligase.

Article Title: Enzyme-guided DNA Sewing Architecture
Article Snippet: Different amounts of Y-DNAs (0.3 μM, 0.6 μM, 1.2 μM and 1.5 μM) were combined with T4 ligase (2 μl) and 10× ligase buffer(5 μl) in a total volume of 50 μl.

Article Title: Enzyme-guided DNA Sewing Architecture
Article Snippet: Specifically, 0.6 μM Y-DNAs were combined with T4 ligase (2 μl) and 10× ligase buffer (5 μl) in a total volume of 50 μl and reacted for 3 hours at 25 °C, overnight at 4 °C or for 1 hour at 37 °C as suggested in a user’s manual.

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.

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). ..

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). ..

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  • 99
    Promega t4 dna ligase
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Promega
    Average 99 stars, based on 578 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-05
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      Buy from Supplier

    93
    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: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/Promega
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-05
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    Image Search Results


    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Journal: Nucleic Acids Research

    Article Title: The role of DNA dependent protein kinase in synapsis of DNA ends

    doi: 10.1093/nar/gkg889

    Figure Lengend Snippet: Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Article Snippet: First, we used AMP-PNP, an ATP analog that supports activity of T4 DNA ligase, but cannot function as a cofactor for DNA-PKCS (Fig. A).

    Techniques: Ligation, Labeling, Incubation, Agarose Gel Electrophoresis

    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

    DNA cleavage is coordinated at 3′Dβ and Jβ RSSs. (A) Schematic of the Jβ1 ω , Jβ1 M2 , and Jβ1 M4 alleles as described in the legend to Fig. 1 with the BW linker ligated to cleaved 3′ Dβ1 and Jβ1.2 signal ends. The position of the oligonucleotide primers used to amplify linker ligated to the 3′ Dβ1 (BW-1H, 3A, and 3B) and Jβ1.2 (BWJ, JA, and JB) signal ends are shown as is the position of the oligonucleotide (P1) used to probe these PCR products. Genomic DNA from Jβ1 M4/ω or Jβ1 M2/ω DN thymocytes was incubated with the BW linker in the presence (+) or absence (−) or T4 DNA ligase and heminested LMPCRs performed to detect 3′ Dβ1 (B) and Jβ1.2 (C) signal ends as described in the Materials and Methods section. Analyses of Jβ1.2 signal ends was performed on ligated thymocyte DNA that was serially diluted into nonligated DNA keeping the total amount of DNA constant. Cell equivalents of ligated template DNA are indicated. Products from ligation of the BW linker to signal ends from the Jβ1 ω (ω), Jβ1 M4 (M4), and Jβ1 M2 (M2) alleles are indicated. Molecular weight markers are shown. Also shown is a RAG-2 gene PCR performed on ligated and nonligated template DNA and probed with the R2P oligonucleotide.

    Journal: The Journal of Experimental Medicine

    Article Title: Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

    doi: 10.1084/jem.20011803

    Figure Lengend Snippet: DNA cleavage is coordinated at 3′Dβ and Jβ RSSs. (A) Schematic of the Jβ1 ω , Jβ1 M2 , and Jβ1 M4 alleles as described in the legend to Fig. 1 with the BW linker ligated to cleaved 3′ Dβ1 and Jβ1.2 signal ends. The position of the oligonucleotide primers used to amplify linker ligated to the 3′ Dβ1 (BW-1H, 3A, and 3B) and Jβ1.2 (BWJ, JA, and JB) signal ends are shown as is the position of the oligonucleotide (P1) used to probe these PCR products. Genomic DNA from Jβ1 M4/ω or Jβ1 M2/ω DN thymocytes was incubated with the BW linker in the presence (+) or absence (−) or T4 DNA ligase and heminested LMPCRs performed to detect 3′ Dβ1 (B) and Jβ1.2 (C) signal ends as described in the Materials and Methods section. Analyses of Jβ1.2 signal ends was performed on ligated thymocyte DNA that was serially diluted into nonligated DNA keeping the total amount of DNA constant. Cell equivalents of ligated template DNA are indicated. Products from ligation of the BW linker to signal ends from the Jβ1 ω (ω), Jβ1 M4 (M4), and Jβ1 M2 (M2) alleles are indicated. Molecular weight markers are shown. Also shown is a RAG-2 gene PCR performed on ligated and nonligated template DNA and probed with the R2P oligonucleotide.

    Article Snippet: Purified thymocyte genomic DNA (2–3 μg) was ligated to 100 pmoles of the BW linker in a volume of 50–60 μl with 1–3 units T4 DNA Ligase (Promega) at 16°C for 12–14 h. Ligated samples were extracted with phenol and chloroform before PCR analysis.

    Techniques: Polymerase Chain Reaction, Incubation, Ligation, Molecular Weight

    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