t4 ligase Roche Search Results


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  • 99
    New England Biolabs t4 dna ligase
    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of <t>T4</t> DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher topo ta cloning kit
    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of <t>T4</t> DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 dna ligase
    Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the <t>T4</t> DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega t4 dna ligase
    Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the <t>T4</t> DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 11518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 polynucleotide kinase
    Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the <t>T4</t> DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9937 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polynucleotide kinase
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher biotin 14 datp
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Biotin 14 Datp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick gel extraction kit
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Qiaquick Gel Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 113321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick pcr purification kit
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript first strand cdna synthesis kit
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Primescript First Strand Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna polymerase i
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaprep spin miniprep kit
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Qiaprep Spin Miniprep Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 42567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dpnii
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Dpnii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1099 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher clonejet pcr cloning kit
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Clonejet Pcr Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher biotin 14 dctp
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    Biotin 14 Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 rna ligase
    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by <t>T4</t> RNA ligase (T4 ligase).
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3749 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase
    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by <t>T4</t> RNA ligase (T4 ligase).
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 ligase
    Positive and negative controls for chromosome conformation capture. All possible restriction fragments after  Bgl II  digestions were present in equimolar amounts. A. Two regions of calreticulin gene,  CalR2.8  and  CalR4.4 , were cross-linked and represented a positive control. On the other hand,  COX4i1  and  CalR  were two functionally unrelated genes, and they did not interact in our 3C reactions. B.  CalR2.8  and  CalR4.4 , as well as  COX4i1  or  COX8a  and  Grin1 ,  Gria2 , or  Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.
    T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bamhi
    (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The <t>BamHI</t> and <t>SalI</t> restriction sites used to clone TH are unique in the vector
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion high fidelity dna polymerase
    (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The <t>BamHI</t> and <t>SalI</t> restriction sites used to clone TH are unique in the vector
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion dna polymerase
    (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The <t>BamHI</t> and <t>SalI</t> restriction sites used to clone TH are unique in the vector
    Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna polymerase
    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with <t>T4</t> DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs antarctic phosphatase
    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with <t>T4</t> DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Antarctic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Journal: BMC Molecular Biology

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles

    doi: 10.1186/s12867-017-0083-2

    Figure Lengend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Article Snippet: Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA).

    Techniques: Methylation, Ligation, Polymerase Chain Reaction, Amplification

    Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the T4 DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.

    Journal: Nucleic Acids Research

    Article Title: Chromatin loop organization of the junb locus in mouse dendritic cells

    doi: 10.1093/nar/gkt669

    Figure Lengend Snippet: Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the T4 DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.

    Article Snippet: In all, 25 μl of this PCR product were digested with Dde I and ligated using T4 DNA ligase (Invitrogen) before a second digestion with Eco RI.

    Techniques: Activity Assay, Transfection, Clone Assay, Luciferase, Plasmid Preparation, Purification

    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Journal: Genome Research

    Article Title: Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

    doi: 10.1101/gr.121426.111

    Figure Lengend Snippet: Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Article Snippet: Total RNA was isolated using TRIzol (Invitrogen), and small RNA samples were 5′ labeled by first treating with calf alkaline phosphatase (Roche) followed by phosphorylation with T4 polynucleotide kinase (NEB) and 32 P-γ-ATP.

    Techniques: Labeling, Molecular Weight

    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Journal: RNA

    Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

    doi: 10.1261/rna.064501.117

    Figure Lengend Snippet: cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Article Snippet: For 5′-end labeling, degraded RNA fragments, γ-P32 -ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32 ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min.

    Techniques: Labeling, Marker

    Positive and negative controls for chromosome conformation capture. All possible restriction fragments after  Bgl II  digestions were present in equimolar amounts. A. Two regions of calreticulin gene,  CalR2.8  and  CalR4.4 , were cross-linked and represented a positive control. On the other hand,  COX4i1  and  CalR  were two functionally unrelated genes, and they did not interact in our 3C reactions. B.  CalR2.8  and  CalR4.4 , as well as  COX4i1  or  COX8a  and  Grin1 ,  Gria2 , or  Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.

    Journal: Journal of neurochemistry

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons

    doi:

    Figure Lengend Snippet: Positive and negative controls for chromosome conformation capture. All possible restriction fragments after Bgl II digestions were present in equimolar amounts. A. Two regions of calreticulin gene, CalR2.8 and CalR4.4 , were cross-linked and represented a positive control. On the other hand, COX4i1 and CalR were two functionally unrelated genes, and they did not interact in our 3C reactions. B. CalR2.8 and CalR4.4 , as well as COX4i1 or COX8a and Grin1 , Gria2 , or Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.

    Article Snippet: Chromatin re-ligation was performed by incubating diluted chromatin with 100 Weiss units of T4 ligase (Fermentas) for 4 h at 16°C.

    Techniques: Positive Control

    (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The BamHI and SalI restriction sites used to clone TH are unique in the vector

    Journal: Protein expression and purification

    Article Title: Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

    doi: 10.1016/j.pep.2012.05.007

    Figure Lengend Snippet: (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The BamHI and SalI restriction sites used to clone TH are unique in the vector

    Article Snippet: Restriction enzymes SalI (cat# R0138S) and BamHI (cat# R0136S), T4 ligase (cat# M0202S), amylose resin (cat# E8021L) were obtained from New England Biolabs (Ipswich, MA).

    Techniques: Construct, Recombinant, Sequencing, Plasmid Preparation

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Journal: PLoS ONE

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    doi: 10.1371/journal.pone.0111538

    Figure Lengend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Article Snippet: One microlitre of dTTP (20 mM; 1.7 mM final concentration) was added to each tube containing the Exo-SAP treated inserts, mixed well by mild vortexing followed by brief centrifugation at 4°C, and 1.5 units (0.5 µl) of T4 DNA polymerase (New England Biolabs) were added.

    Techniques: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation