splintr ligase  (New England Biolabs)


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    Name:
    SplintR Ligase
    Description:
    SplintR Ligase 6 250 units
    Catalog Number:
    M0375L
    Price:
    422
    Size:
    6 250 units
    Category:
    DNA Ligases
    Score:
    85
    Buy from Supplier


    Structured Review

    New England Biolabs splintr ligase
    SplintR Ligase
    SplintR Ligase 6 250 units
    https://www.bioz.com/result/splintr ligase/product/New England Biolabs
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    splintr ligase - by Bioz Stars, 2019-11
    93/100 stars

    Images

    1) Product Images from "Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation"

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation

    Journal: eLife

    doi: 10.7554/eLife.03700

    Examination of SplintR ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007
    Figure Legend Snippet: Examination of SplintR ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007

    Techniques Used: Ligation

    Related Articles

    Amplification:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Therefore, in the next step, optimization of the RNase H concentration was necessary for RNase H-assisted RPRCA. .. After hybridization and ligation of the padlock probe by SplintR ligase, RNase H-assisted RPRCA was performed in the absence or presence of various concentrations of RNase H. Initially the resulting amplified ssDNA was analyzed by agarose gel electrophoresis. .. We found that ssDNA products did not arise in the absence of both GFP mRNA and the padlock probe or in the presence of only GFP mRNA or the padlock probe.

    Agarose Gel Electrophoresis:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Therefore, in the next step, optimization of the RNase H concentration was necessary for RNase H-assisted RPRCA. .. After hybridization and ligation of the padlock probe by SplintR ligase, RNase H-assisted RPRCA was performed in the absence or presence of various concentrations of RNase H. Initially the resulting amplified ssDNA was analyzed by agarose gel electrophoresis. .. We found that ssDNA products did not arise in the absence of both GFP mRNA and the padlock probe or in the presence of only GFP mRNA or the padlock probe.

    In Vitro:

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation
    Article Snippet: The template sequence for the initial ligase screen ( ) was a 307 nt section of mouse DDX1 mRNA (NM_134040.1; see ); ssDNA and RNA templates were a synthetic oligonucleotide and in vitro transcript, respectively. .. Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB).

    Fluorescence:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: T4Dnl could only seal the RNA-splinted padlock probe under low ATP concentrations (10 µM) as reported previously (Fig. ) – . .. A comparison of the fluorescence intensities of the circular ssDNA probes suggested that the sealing efficiency of T4Dnl is lower than that of SplintR ligase in the presence of 400 µM ATP (Fig. ). .. However, an obvious circularized padlock probe band was often not confirmed (data not shown).

    Ligation:

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation
    Article Snippet: Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB). .. Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB).

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Therefore, in the next step, optimization of the RNase H concentration was necessary for RNase H-assisted RPRCA. .. After hybridization and ligation of the padlock probe by SplintR ligase, RNase H-assisted RPRCA was performed in the absence or presence of various concentrations of RNase H. Initially the resulting amplified ssDNA was analyzed by agarose gel electrophoresis. .. We found that ssDNA products did not arise in the absence of both GFP mRNA and the padlock probe or in the presence of only GFP mRNA or the padlock probe.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Recently, it was demonstrated that the ATP-dependent DNA ligase of chlorella virus PBCV-1 , which is commercially available as SplintR ligase (New England BioLabs, Ipswich, MA, USA), can seal RNA-splinted DNA with high efficiency . .. In this study, we developed a novel RPRCA procedure for the direct detection of mRNA molecules in combination with RNase H, a padlock DNA probe, and SplintR ligase, which can prepare a circular probe from an RNA-splinted padlock DNA probe with higher ligation efficiency than T4Rnl2 and T4Dnl. .. This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule.

    Produced:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: In the present study, we developed a simple method for the direct detection of sequences of interest in target RNA molecules without RT. .. RNase H specifically recognized the target RNA hybridized with a circularized ssDNA probe, which was produced using an RNA-splinted padlock probe and SplintR ligase. .. Our first concern in this study was that the signal-to-noise ratio worsened due to background DNA synthesis from non-circularized padlock probes, as we reported previously , .

    Concentration Assay:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: T4Dnl can recognize an RNA-splinted padlock probe as a substrate by reducing the adenosine triphosphate (ATP) concentration in the reaction mixture to 10 µM – , although the sealing efficiency of T4Dnl is significantly lower for RNA-splinted padlock probes than for ‘DNA-splinted padlock probes’ , . .. Recently, it was demonstrated that the ATP-dependent DNA ligase of chlorella virus PBCV-1 , which is commercially available as SplintR ligase (New England BioLabs, Ipswich, MA, USA), can seal RNA-splinted DNA with high efficiency .

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: We found that irrespective of the ATP concentration, a band corresponding to the circular ssDNA probe was observable in the presence of SprintR ligase (Fig. ). .. The yield of the circularized padlock probe by SplintR ligase was stable under all ATP concentration conditions (10 µM, 62.8 ± 4.2%; 400 µM, 67.5 ± 1.1%; and 1 mM, 61.9 ± 3.4%; Fig. ). .. T4Dnl could only seal the RNA-splinted padlock probe under low ATP concentrations (10 µM) as reported previously (Fig. ) – .

    Incubation:

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation
    Article Snippet: Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB). .. Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB).

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Hybridization of the padlock probe was facilitated by incubation at 95 °C for 1 min followed by immediate cooling to 40 °C and incubation for 10 min at 30 °C. .. After hybridization, 1 µl of SplintR ligase (25 units) was added to the reaction mixture, which was incubated at 37 °C for 15 min to seal the padlock probe. .. The RNase H-assisted RPRCA reaction was started by mixing 10 µl of the ligated mixture with 10 µl of a reaction mixture containing 20 mM Tris-acetate (pH 7.5), 10 mM MgAc, 80 mM ammonium sulfate ((NH4 )2 SO4 ), 10 mM KGlu, 2.0 mM deoxynucleoside triphosphate, 10 mM dithiothreitol, 0.002 units of pyrophosphatase (New England BioLabs), an appropriate number of units of RNase H (Figs – , BioAcademia, Ibaraki, Osaka, Japan), and 100 ng of DNA-free phi29 DNA polymerase (Kanto Chemical).

    other:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: By contrast, T4Dnl and SplintR ligase could seal RNA-splinted padlock probes, and SplintR ligase was more suitable for this process.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: We found that all padlock probes were converted into circular ssDNA by SplintR ligase (Fig. ).

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: This experiment clearly illustrated that SplintR ligase is most suitable for the circularization of RNA-splinted padlock probes among the ligases tested.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: In addition, as observed previously , the sealing efficiency of SplintR ligase was affected by the nucleotide pair at both arm ends of the padlock probe.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: RNase H cannot convert “DNA-splint” into a primer; therefore, our procedure using RNase H can detect only target RNA molecules even if SplintR ligase can also seal the DNA-splinted padlock probe.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Therefore, we first examined whether SplintR ligase can more efficiently seal an RNA-splinted padlock probe than T4Dnl and T4Rnl2, which were used previously – , – .

    Activity Assay:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: However, its activity has not been confirmed by other research groups , , – ; thus, the sealing activity of T4Rnl2 regarding RNA-splinted padlock probes remains controversial. .. Recently, it was demonstrated that the ATP-dependent DNA ligase of chlorella virus PBCV-1 , which is commercially available as SplintR ligase (New England BioLabs, Ipswich, MA, USA), can seal RNA-splinted DNA with high efficiency .

    RNA Detection:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Similarly, regarding RNA detection, two ligases, namely T4 DNA ligase (T4Dnl) and T4 RNA ligase 2 (T4Rnl2), can be used to seal an ‘RNA-splinted padlock probe’. .. Recently, it was demonstrated that the ATP-dependent DNA ligase of chlorella virus PBCV-1 , which is commercially available as SplintR ligase (New England BioLabs, Ipswich, MA, USA), can seal RNA-splinted DNA with high efficiency .

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule. .. Figure shows a basic scheme for target RNA detection by RNase H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi29 DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) finally, RPRCA synthesizes long ssDNA, which has a periodic sequence complementary to the padlock probe. .. In our strategy, both the detection sensitivity and specificity depend on the sealing efficiency of the RNA-splinted padlock probe.

    Sequencing:

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation
    Article Snippet: The template sequence for the initial ligase screen ( ) was a 307 nt section of mouse DDX1 mRNA (NM_134040.1; see ); ssDNA and RNA templates were a synthetic oligonucleotide and in vitro transcript, respectively. .. Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB).

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: In addition, as observed previously , the sealing efficiency of SplintR ligase was affected by the nucleotide pair at both arm ends of the padlock probe. .. Therefore, we examined whether the padlock probe position and arm sequence on the target mRNA affected the sealing efficiency of SplintR ligase. .. The arm positions and nucleotide pair at both arm ends of each padlock probe on the target GFP mRNA are illustrated in Fig. , and the chemical characteristics of each padlock probe (e.g., the melting temperature [Tm] of the arm position) used in this study are also shown in Supplementary Table .

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule. .. Figure shows a basic scheme for target RNA detection by RNase H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi29 DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) finally, RPRCA synthesizes long ssDNA, which has a periodic sequence complementary to the padlock probe. .. In our strategy, both the detection sensitivity and specificity depend on the sealing efficiency of the RNA-splinted padlock probe.

    DNA Synthesis:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule. .. Figure shows a basic scheme for target RNA detection by RNase H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi29 DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) finally, RPRCA synthesizes long ssDNA, which has a periodic sequence complementary to the padlock probe. .. In our strategy, both the detection sensitivity and specificity depend on the sealing efficiency of the RNA-splinted padlock probe.

    Hybridization:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Hybridization of the padlock probe was facilitated by incubation at 95 °C for 1 min followed by immediate cooling to 40 °C and incubation for 10 min at 30 °C. .. After hybridization, 1 µl of SplintR ligase (25 units) was added to the reaction mixture, which was incubated at 37 °C for 15 min to seal the padlock probe. .. The RNase H-assisted RPRCA reaction was started by mixing 10 µl of the ligated mixture with 10 µl of a reaction mixture containing 20 mM Tris-acetate (pH 7.5), 10 mM MgAc, 80 mM ammonium sulfate ((NH4 )2 SO4 ), 10 mM KGlu, 2.0 mM deoxynucleoside triphosphate, 10 mM dithiothreitol, 0.002 units of pyrophosphatase (New England BioLabs), an appropriate number of units of RNase H (Figs – , BioAcademia, Ibaraki, Osaka, Japan), and 100 ng of DNA-free phi29 DNA polymerase (Kanto Chemical).

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Therefore, in the next step, optimization of the RNase H concentration was necessary for RNase H-assisted RPRCA. .. After hybridization and ligation of the padlock probe by SplintR ligase, RNase H-assisted RPRCA was performed in the absence or presence of various concentrations of RNase H. Initially the resulting amplified ssDNA was analyzed by agarose gel electrophoresis. .. We found that ssDNA products did not arise in the absence of both GFP mRNA and the padlock probe or in the presence of only GFP mRNA or the padlock probe.

    In Situ:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Furthermore, our results revealed that RNase H was more effective for RNA sequence detection in RPRCA even though phi29 DNA polymerase has 3′ to 5′ exoribonuclease activity. .. Two recent papers , reported that in situ RCA can detect eukaryotic mRNA using a padlock probe and SplintR ligase. .. Previously, RCA for detecting eukaryotic mRNA was performed after RT using target-specific primers .

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Previously, RCA for detecting eukaryotic mRNA was performed after RT using target-specific primers . .. Therefore, this method combining a padlock probe with SplintR ligase enabled the RCA reaction for in situ mRNA detection without RT. .. Thus, RNase H-assisted RPRCA would be also suitable for in situ mRNA detection even if the RNA has a poly(A) tail.

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  • 93
    New England Biolabs splintr ligase
    Examination of <t>SplintR</t> ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007
    Splintr Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/splintr ligase/product/New England Biolabs
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    splintr ligase - by Bioz Stars, 2019-11
    93/100 stars
      Buy from Supplier

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    Examination of SplintR ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007

    Journal: eLife

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation

    doi: 10.7554/eLife.03700

    Figure Lengend Snippet: Examination of SplintR ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007

    Article Snippet: Enzymes tested were Tth DNA ligase (AB-0325; Thermo, Waltham, MA), Tsc DNA ligase (Dlig 119; Prokaria, Reykjavik, Iceland), thermostable DNA ligase (BIO-27045; Bioline, Taunton, MA), T4 DNA ligase (M0202S; NEB, Ipswich, MA), Escherichia coli DNA ligase (M0205S; NEB), Rnl2 (M0239; NEB), SplintR ligase (M0375; NEB).

    Techniques: Ligation