t4 rna ligase 1 Search Results


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  • 95
    New England Biolabs t4 rna ligase 1
    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using <t>T4</t> RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.
    T4 Rna Ligase 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Jena Bioscience pcp cy3
    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using <t>T4</t> RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.
    Pcp Cy3, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Illumina Inc t4 rna ligase 1
    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using <t>T4</t> RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.
    T4 Rna Ligase 1, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 96/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Enzymatics t4 rna ligase 1
    Adenylation of 3’ adapter using <t>T4</t> RNA ligase 1. ( A ) Effect of PEG8000 concentration on adenylation efficiency. A synthetic oligo BL1 mimicking the first small RNA cloning linker reported by Lau et al. (2001) was adenylated overnight with 1 U/μL T4 RNA ligase at various PEG concentration. Non-adenylated oligo as the negative control (NC) is loaded on the left lane. ( B ) Effect of temperature and 5’ nucleotide composition on adenylation efficiency. Oligos were adenylated overnight in the presence of 20% PEG8000 at various temperatures. ( C ) Impact of oligo concentration on adenylation efficiency. Substrates with different concentrations were adenylated overnight with 20% PEG8000 at room temperature. All adenylation products were analyzed on the 20% denatured PAGE, stained with SYBR-Gold and photograph under UV.
    T4 Rna Ligase 1, supplied by Enzymatics, used in various techniques. Bioz Stars score: 96/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Epicentre Biotechnologies t4 rnl1
    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or <t>T4</t> Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    T4 Rnl1, supplied by Epicentre Biotechnologies, used in various techniques. Bioz Stars score: 79/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs m0201s l t4 rna ligase 1
    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or <t>T4</t> Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    M0201s L T4 Rna Ligase 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 rna ligase
    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by <t>T4</t> RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t4 rna ligase 1 reaction buffer
    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by <t>T4</t> RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    T4 Rna Ligase 1 Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 1 reaction buffer/product/New England Biolabs
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    95
    New England Biolabs t4 rna ligase 1 t4 rna ligase 2
    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by <t>T4</t> RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    T4 Rna Ligase 1 T4 Rna Ligase 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t4 rna ligase
    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by <t>T4</t> RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2987 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/New England Biolabs
    Average 95 stars, based on 2987 article reviews
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    90
    TaKaRa t4 rna ligase
    Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with <t>T4</t> RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.
    T4 Rna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Jena Bioscience pcp cy5
    Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with <t>T4</t> RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.
    Pcp Cy5, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Promega t4 rna ligase
    Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with <t>T4</t> RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.
    T4 Rna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 626 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    T4 RNA Ligase 1 catalyzes the ATP dependent ligation of 5 phosphoryl terminated nucleic acid donor to a 3 hydroxyl terminated nucleic acid acceptor through the formation of a 3
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    Image Search Results


    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Purification, Modification, Incubation

    microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Ligation, Generated, Labeling

    Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Generated, Ligation, DNA Ligation

    Adenylation of 3’ adapter using T4 RNA ligase 1. ( A ) Effect of PEG8000 concentration on adenylation efficiency. A synthetic oligo BL1 mimicking the first small RNA cloning linker reported by Lau et al. (2001) was adenylated overnight with 1 U/μL T4 RNA ligase at various PEG concentration. Non-adenylated oligo as the negative control (NC) is loaded on the left lane. ( B ) Effect of temperature and 5’ nucleotide composition on adenylation efficiency. Oligos were adenylated overnight in the presence of 20% PEG8000 at various temperatures. ( C ) Impact of oligo concentration on adenylation efficiency. Substrates with different concentrations were adenylated overnight with 20% PEG8000 at room temperature. All adenylation products were analyzed on the 20% denatured PAGE, stained with SYBR-Gold and photograph under UV.

    Journal: Plant Methods

    Article Title: A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

    doi: 10.1186/1746-4811-8-41

    Figure Lengend Snippet: Adenylation of 3’ adapter using T4 RNA ligase 1. ( A ) Effect of PEG8000 concentration on adenylation efficiency. A synthetic oligo BL1 mimicking the first small RNA cloning linker reported by Lau et al. (2001) was adenylated overnight with 1 U/μL T4 RNA ligase at various PEG concentration. Non-adenylated oligo as the negative control (NC) is loaded on the left lane. ( B ) Effect of temperature and 5’ nucleotide composition on adenylation efficiency. Oligos were adenylated overnight in the presence of 20% PEG8000 at various temperatures. ( C ) Impact of oligo concentration on adenylation efficiency. Substrates with different concentrations were adenylated overnight with 20% PEG8000 at room temperature. All adenylation products were analyzed on the 20% denatured PAGE, stained with SYBR-Gold and photograph under UV.

    Article Snippet: Reagents T4 RNA liagse 2, truncated (NEB) 50% PEG8000 (NEB) 10 mM ATP (NEB) RNase Inhibitor (Promega) T4 RNA ligase 1 (Enzymatics) SuperScriptIII Reverse Transcriptase (Invitrogen) Phusion Hotstart DNA Polymerase (NEB) MinElute Gel Extraction kit (Qiagen) Quant-iT HS DNA assay kit (Inivtrogen)

    Techniques: Concentration Assay, Clone Assay, Negative Control, Polyacrylamide Gel Electrophoresis, Staining

    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Journal: RNA

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    doi: 10.1261/rna.2242610

    Figure Lengend Snippet: RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Article Snippet: Condition 1 was 1 unit of T4 Rnl1 (Epicentre Biotechnology) in 1× reaction buffer without ATP (33 mM Tris-acetate at pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, and 0.5 mM DTT) containing 25% (w/v) PEG.

    Techniques: Ligation, Staining

    RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM;  n  = 3 experimental replicates.

    Journal: RNA

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    doi: 10.1261/rna.2242610

    Figure Lengend Snippet: RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Article Snippet: Condition 1 was 1 unit of T4 Rnl1 (Epicentre Biotechnology) in 1× reaction buffer without ATP (33 mM Tris-acetate at pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, and 0.5 mM DTT) containing 25% (w/v) PEG.

    Techniques: Ligation, Methylation, Staining

    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Journal: The Analyst

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    doi: 10.1039/c7an00670e

    Figure Lengend Snippet: Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Article Snippet: Specifically, 1 nmole of 5′-phosphorylated 3′ adaptor was mixed with 40 μl 50% PEG 8000 (BioLabs), 10 μl 1x T4 RNA ligase reaction buffer, and 5 μl T4 RNA ligase 1 (Thermo Fisher EL0021) in a total volume of 100 μl and reacted at room temperature overnight.

    Techniques: Lysis, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Selection, Gel Purification

    Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with T4 RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.

    Journal: Biological Procedures Online

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

    doi: 10.1251/bpo33

    Figure Lengend Snippet: Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with T4 RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.

    Article Snippet: Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC.

    Techniques: Nucleic Acid Electrophoresis, Ligation, Polyacrylamide Gel Electrophoresis, Fluorescence, Staining, SYBR Green Assay

    Time course of ligation reactions. Concentrations were [mRNA] = 0.5 µM and [T4 RNA ligase] = 1 U/µl in the enzyme-supplier's buffer with 10% DMSO. At 25 ºC.(a) Y-ligation, [mRNA]:[Linker-Y] = 1:1.5(b) Splint ligation with T4 RNA ligase as compared with Y-ligation, [mRNA]:[Splint]:[Linker-S] = 1:1:1.5

    Journal: Biological Procedures Online

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

    doi: 10.1251/bpo33

    Figure Lengend Snippet: Time course of ligation reactions. Concentrations were [mRNA] = 0.5 µM and [T4 RNA ligase] = 1 U/µl in the enzyme-supplier's buffer with 10% DMSO. At 25 ºC.(a) Y-ligation, [mRNA]:[Linker-Y] = 1:1.5(b) Splint ligation with T4 RNA ligase as compared with Y-ligation, [mRNA]:[Splint]:[Linker-S] = 1:1:1.5

    Article Snippet: Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC.

    Techniques: Ligation