pnk buffer  (New England Biolabs)


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    New England Biolabs pnk buffer
    Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pnk buffer/product/New England Biolabs
    Average 99 stars, based on 63 article reviews
    Price from $9.99 to $1999.99
    pnk buffer - by Bioz Stars, 2020-01
    99/100 stars

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    Centrifugation:

    Article Title: Dynamic Repositioning of Dorsal to Two Different ?B Motifs Controls Its Autoregulation during Immune Response in Drosophila
    Article Snippet: Embryonic nuclear extracts were prepared by homogenizing embryos (2–4-h-old) in extraction buffer (20 m m Hepes, pH 7.9, 5 m m MgCl2 , 0.1 m m EGTA, 12.5% sucrose, 25% glycerol, 0.5 m m dithiothreitol, 0.5 m m phenylmethylsulfonyl fluoride and protease inhibitor mixture) using a Dounce homogenizer, followed by centrifugation at 3000 × g for 15 min at 4 °C. .. For EMSA, 100 ng each of different double-stranded oligonucleotide probes was labeled with 2 μl of [γ-32 P]ATP (5 × 105 cpm) and 1 μl of polynucleotide kinase (10 units/μl) in 1 μl of PNK buffer (New England Biolabs) for 1 h at 37 °C.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts
    Article Snippet: Formamide loading buffer: Gel loading buffer II for denaturing PAGE (Ambion): 95% Formamide, 18 mM EDTA, and 0.025 % SDS, 0.025% Xylene Cyanol, and 0.025% Bromophenol Blue. .. 10 × PNK buffer (NEB) Thermo Sequenase Cycle Sequencing Kit (USB/Affimetrix). pControl (250 ng/μl).

    Electrophoresis:

    Article Title: DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2
    Article Snippet: The oligonucleotide probe, which was 32 P-labeled at the 5'-terminus (2 nM, molecules), was added to the diluted mixture, as well as PNK buffer (New England Biolabs) to final concentrations of 7 mM Tris-HCl (pH 7.6), 1 mM MgCl2 , and 0.5 mM dithiothreitol. .. The products were then separated by electrophoresis in 1% agarose and analyzed by Storm 860 PhosphorImager (GE Healthcare).

    Incubation:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: .. The beads were washed three times with PNK Buffer and incubated in 80 μl PNK Buffer (NEB) with 40 units of T4 PNK enzyme (NEB) in the presence of 32 P-γ-ATP (1 mCi). .. Cold ATP was added to the reaction at a final concentration of 1.25 mM and incubated for 5 min.

    Article Title: Automethylation of CARM1 allows coupling of transcription and mRNA splicing
    Article Snippet: .. 32 P-labeled PCR The forward primer for the CT/CGRP mini-gene was labeled by incubation with polynucleotide kinase (PNK) in PNK buffer (New England Biolabs) with γ-labeled 32 P-ATP at a final concentration of 1 µM per primer. .. Reactions were held at 37°C for 30 min. PCR was performed with Taq polymerase (Promega), 2 µl of 1:100 diluted cDNA per 10 µl reaction and a final primer concentration of 600 nM.

    Article Title: C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1
    Article Snippet: .. C6 pyridinium ceramide treatment of HeLa nuclear extracts Ten micrograms of HeLa nuclear extract (NE) was incubated with 4 µCi [γ32 P] ATP for 30 min at 37°C in 1× PNK buffer (New England Bio Labs) plus or 10 µM C6 pyridinium ceramide in a 50 µl final volume. .. Radiolabeled-NE proteins were then subjected to immunoprecipitation overnight with 2 µg of indicated antibodies, 10 µl proteins A/G beads (Santa Cruz) and 200 µl RIPA-rescue buffer containing phosphatase and protease inhibitors.

    Diffusion-based Assay:

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: In vitro transcription, RNA cleaning, dephosphorylation and labeling was carried out according to ( ) with minor deviations: RNA was extracted from gel by simple diffusion into 2 M NH4 -acetate without electro elution. .. Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB).

    Modification:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: An RNA linker (5'- CUCGUAUGCCGUCUUCUGCUUG-3’ 3’ Puromycin, 5’P) with a puromycin modification at the 3′ end to avoid self-circularization was linked to the mRNA present in the complexes by T4 RNA Ligase and incubated overnight at 16°C with gentle shaking (1300 rpm every 5 min for 15 s in the Thermomixer). .. The beads were washed three times with PNK Buffer and incubated in 80 μl PNK Buffer (NEB) with 40 units of T4 PNK enzyme (NEB) in the presence of 32 P-γ-ATP (1 mCi).

    Hybridization:

    Article Title: Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication
    Article Snippet: Prior to hybridization with the radio-labeled DNA probe, the membrane was kept at 4°C. .. The radio-labeled DNA probe was prepared by mixing 1 µl of 25 µM antisense DNA probe with 6 µl of nuclease-free H2 O and 1 µl of 10× PNK buffer (New England Biolabs), then heating at 100°C for 2 min, and placing on ice immediately.

    Article Title: DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2
    Article Snippet: To analyze the directionality of resection by hybridization with radiolabeled oligonucleotide probes (as in ), a standard reaction was first carried out as described above. .. The oligonucleotide probe, which was 32 P-labeled at the 5'-terminus (2 nM, molecules), was added to the diluted mixture, as well as PNK buffer (New England Biolabs) to final concentrations of 7 mM Tris-HCl (pH 7.6), 1 mM MgCl2 , and 0.5 mM dithiothreitol.

    Concentration Assay:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: The beads were washed three times with PNK Buffer and incubated in 80 μl PNK Buffer (NEB) with 40 units of T4 PNK enzyme (NEB) in the presence of 32 P-γ-ATP (1 mCi). .. Cold ATP was added to the reaction at a final concentration of 1.25 mM and incubated for 5 min.

    Article Title: Automethylation of CARM1 allows coupling of transcription and mRNA splicing
    Article Snippet: .. 32 P-labeled PCR The forward primer for the CT/CGRP mini-gene was labeled by incubation with polynucleotide kinase (PNK) in PNK buffer (New England Biolabs) with γ-labeled 32 P-ATP at a final concentration of 1 µM per primer. .. Reactions were held at 37°C for 30 min. PCR was performed with Taq polymerase (Promega), 2 µl of 1:100 diluted cDNA per 10 µl reaction and a final primer concentration of 600 nM.

    Protease Inhibitor:

    Article Title: Dynamic Repositioning of Dorsal to Two Different ?B Motifs Controls Its Autoregulation during Immune Response in Drosophila
    Article Snippet: Embryonic nuclear extracts were prepared by homogenizing embryos (2–4-h-old) in extraction buffer (20 m m Hepes, pH 7.9, 5 m m MgCl2 , 0.1 m m EGTA, 12.5% sucrose, 25% glycerol, 0.5 m m dithiothreitol, 0.5 m m phenylmethylsulfonyl fluoride and protease inhibitor mixture) using a Dounce homogenizer, followed by centrifugation at 3000 × g for 15 min at 4 °C. .. For EMSA, 100 ng each of different double-stranded oligonucleotide probes was labeled with 2 μl of [γ-32 P]ATP (5 × 105 cpm) and 1 μl of polynucleotide kinase (10 units/μl) in 1 μl of PNK buffer (New England Biolabs) for 1 h at 37 °C.

    Northern Blot:

    Article Title: Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication
    Article Snippet: Paragraph title: Northern Blot analysis ... The radio-labeled DNA probe was prepared by mixing 1 µl of 25 µM antisense DNA probe with 6 µl of nuclease-free H2 O and 1 µl of 10× PNK buffer (New England Biolabs), then heating at 100°C for 2 min, and placing on ice immediately.

    Imaging:

    Article Title: DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2
    Article Snippet: Gels were analyzed using an AlphaImager HP (Alpha Innotech) imaging station, and are presented as the inverted image. .. The oligonucleotide probe, which was 32 P-labeled at the 5'-terminus (2 nM, molecules), was added to the diluted mixture, as well as PNK buffer (New England Biolabs) to final concentrations of 7 mM Tris-HCl (pH 7.6), 1 mM MgCl2 , and 0.5 mM dithiothreitol.

    Sequencing:

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts
    Article Snippet: .. 10 × PNK buffer (NEB) Thermo Sequenase Cycle Sequencing Kit (USB/Affimetrix). pControl (250 ng/μl). ..

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Paragraph title: Crosslinking Immunoprecipitation and deep sequencing (CLIP-seq) in C. elegans ... The beads were washed three times with PNK Buffer and incubated in 80 μl PNK Buffer (NEB) with 40 units of T4 PNK enzyme (NEB) in the presence of 32 P-γ-ATP (1 mCi).

    Binding Assay:

    Article Title: Dynamic Repositioning of Dorsal to Two Different ?B Motifs Controls Its Autoregulation during Immune Response in Drosophila
    Article Snippet: For EMSA, 100 ng each of different double-stranded oligonucleotide probes was labeled with 2 μl of [γ-32 P]ATP (5 × 105 cpm) and 1 μl of polynucleotide kinase (10 units/μl) in 1 μl of PNK buffer (New England Biolabs) for 1 h at 37 °C. .. The labeled DNA was purified, and binding reaction was performed for 45 min at room temperature by mixing 1 ng of purified 32 P-labeled double-stranded synthetic oligonucleotide probe (4000 cpm/μl), 10 μl of nuclear extracts, and 300 ng of poly(dI-dC) in the presence of a protease inhibitor mixture (Sigma).

    Isolation:

    Article Title: Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication
    Article Snippet: Northern Blot analysis Twenty micrograms of total RNA isolated from shrimp stomach was heated at 100°C for 5 min for denaturation, and then put on ice immediately for at least 1 min. .. The radio-labeled DNA probe was prepared by mixing 1 µl of 25 µM antisense DNA probe with 6 µl of nuclease-free H2 O and 1 µl of 10× PNK buffer (New England Biolabs), then heating at 100°C for 2 min, and placing on ice immediately.

    Size-exclusion Chromatography:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: The beads were incubated for 10 min at 37°C in the Thermomixer with intermittent shaking (1200 rpm for 15 sec every 3 min) in 80 μl NEB Buffer 3 containing 30 units of Calf Intestinal Phosphatase (NEB). .. The beads were washed three times with PNK Buffer and incubated in 80 μl PNK Buffer (NEB) with 40 units of T4 PNK enzyme (NEB) in the presence of 32 P-γ-ATP (1 mCi).

    Labeling:

    Article Title: Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication
    Article Snippet: The radio-labeled DNA probe was prepared by mixing 1 µl of 25 µM antisense DNA probe with 6 µl of nuclease-free H2 O and 1 µl of 10× PNK buffer (New England Biolabs), then heating at 100°C for 2 min, and placing on ice immediately. .. The probe was labeled by adding 1 µl of T4 polynucleotide kinase (10 U/μl; New England Biolabs) and 1 µl of 32 Pγ-ATP (7,000 Ci/nmole), and then incubating the mixture at 37°C for 2 h. Finally, the mixture was heated at 100°C for 5 min, and passed through a mini Quick Spin™ Oligo Columns (Qiagen) to remove any remaining free isotope.

    Article Title: Automethylation of CARM1 allows coupling of transcription and mRNA splicing
    Article Snippet: .. 32 P-labeled PCR The forward primer for the CT/CGRP mini-gene was labeled by incubation with polynucleotide kinase (PNK) in PNK buffer (New England Biolabs) with γ-labeled 32 P-ATP at a final concentration of 1 µM per primer. .. Reactions were held at 37°C for 30 min. PCR was performed with Taq polymerase (Promega), 2 µl of 1:100 diluted cDNA per 10 µl reaction and a final primer concentration of 600 nM.

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: In vitro transcription, RNA cleaning, dephosphorylation and labeling was carried out according to ( ) with minor deviations: RNA was extracted from gel by simple diffusion into 2 M NH4 -acetate without electro elution. .. Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB).

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: DNA substrates Oligonucleotides were labeled either at the 5’ terminus with [γ-32 P]ATP and T4 polynucleotide kinase (New England Biolabs), or at the 3' terminus with [α-32 P] cordycepin-5-triphosphate and terminal transferase (New England Biolabs) according to standard protocols. .. The substrates were prepared by annealing the 32 P-labeled oligonucleotide with a two-fold excess of the unlabeled oligonucleotide in a PNK buffer (New England Biolabs).

    Article Title: Dynamic Repositioning of Dorsal to Two Different ?B Motifs Controls Its Autoregulation during Immune Response in Drosophila
    Article Snippet: .. For EMSA, 100 ng each of different double-stranded oligonucleotide probes was labeled with 2 μl of [γ-32 P]ATP (5 × 105 cpm) and 1 μl of polynucleotide kinase (10 units/μl) in 1 μl of PNK buffer (New England Biolabs) for 1 h at 37 °C. .. The labeled DNA was purified, and binding reaction was performed for 45 min at room temperature by mixing 1 ng of purified 32 P-labeled double-stranded synthetic oligonucleotide probe (4000 cpm/μl), 10 μl of nuclear extracts, and 300 ng of poly(dI-dC) in the presence of a protease inhibitor mixture (Sigma).

    Purification:

    Article Title: Role of sequence encoded κB DNA geometry in gene regulation by Dorsal
    Article Snippet: A total of 100 ng of double-stranded oligo was labelled with 3 µl of [γ-32 P] ATP and 1 µl of polynucleotide kinase in 1 µl PNK buffer (New England BioLabs) for 1 h at 37°C. .. The labelled DNA was purified on a G50 column.

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB). .. Labeled transcripts were purified before gel shift employing Nucleospin miRNA kit (Macherey–Nagel) according to the manufacturer's instructions.

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: The substrates were prepared by annealing the 32 P-labeled oligonucleotide with a two-fold excess of the unlabeled oligonucleotide in a PNK buffer (New England Biolabs). .. The pUC19 plasmid was digested by HindIII-HF restriction enzyme and purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Dynamic Repositioning of Dorsal to Two Different ?B Motifs Controls Its Autoregulation during Immune Response in Drosophila
    Article Snippet: For EMSA, 100 ng each of different double-stranded oligonucleotide probes was labeled with 2 μl of [γ-32 P]ATP (5 × 105 cpm) and 1 μl of polynucleotide kinase (10 units/μl) in 1 μl of PNK buffer (New England Biolabs) for 1 h at 37 °C. .. The labeled DNA was purified, and binding reaction was performed for 45 min at room temperature by mixing 1 ng of purified 32 P-labeled double-stranded synthetic oligonucleotide probe (4000 cpm/μl), 10 μl of nuclear extracts, and 300 ng of poly(dI-dC) in the presence of a protease inhibitor mixture (Sigma).

    Polymerase Chain Reaction:

    Article Title: Automethylation of CARM1 allows coupling of transcription and mRNA splicing
    Article Snippet: .. 32 P-labeled PCR The forward primer for the CT/CGRP mini-gene was labeled by incubation with polynucleotide kinase (PNK) in PNK buffer (New England Biolabs) with γ-labeled 32 P-ATP at a final concentration of 1 µM per primer. .. Reactions were held at 37°C for 30 min. PCR was performed with Taq polymerase (Promega), 2 µl of 1:100 diluted cDNA per 10 µl reaction and a final primer concentration of 600 nM.

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: The third PCR was carried out on the products of PCR 1 and 2 using primer pair T7_lapB_fw/rev. .. Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB).

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: 10× PNK buffer (NEB): 700 mM Tris-HCl, pH 7.6, 100 mM MgCl2 , 50 mM DTT. .. Proteinase K solution: 0.71 % SDS, 0.357 M EDTA and 4.2 mg/ml Proteinase K (Roche #03115801001, PCR grade).

    Electrophoretic Mobility Shift Assay:

    Article Title: Role of sequence encoded κB DNA geometry in gene regulation by Dorsal
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assay (EMSA) ... A total of 100 ng of double-stranded oligo was labelled with 3 µl of [γ-32 P] ATP and 1 µl of polynucleotide kinase in 1 µl PNK buffer (New England BioLabs) for 1 h at 37°C.

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays (EMSA) ... Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB).

    Article Title: Dynamic Repositioning of Dorsal to Two Different ?B Motifs Controls Its Autoregulation during Immune Response in Drosophila
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assay ... For EMSA, 100 ng each of different double-stranded oligonucleotide probes was labeled with 2 μl of [γ-32 P]ATP (5 × 105 cpm) and 1 μl of polynucleotide kinase (10 units/μl) in 1 μl of PNK buffer (New England Biolabs) for 1 h at 37 °C.

    De-Phosphorylation Assay:

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: .. Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB). .. Labeled transcripts were purified before gel shift employing Nucleospin miRNA kit (Macherey–Nagel) according to the manufacturer's instructions.

    SDS Page:

    Article Title: C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1
    Article Snippet: C6 pyridinium ceramide treatment of HeLa nuclear extracts Ten micrograms of HeLa nuclear extract (NE) was incubated with 4 µCi [γ32 P] ATP for 30 min at 37°C in 1× PNK buffer (New England Bio Labs) plus or 10 µM C6 pyridinium ceramide in a 50 µl final volume. .. Immunoprecipitates were subjected to SDS–PAGE, proteins were transferred on to nitrocellulose membranes and analyzed by phosphor-imaging.

    Plasmid Preparation:

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: The substrates were prepared by annealing the 32 P-labeled oligonucleotide with a two-fold excess of the unlabeled oligonucleotide in a PNK buffer (New England Biolabs). .. The pUC19 plasmid was digested by HindIII-HF restriction enzyme and purified by phenol-chloroform extraction and ethanol precipitation.

    In Vitro:

    Article Title: A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB
    Article Snippet: In vitro transcription, RNA cleaning, dephosphorylation and labeling was carried out according to ( ) with minor deviations: RNA was extracted from gel by simple diffusion into 2 M NH4 -acetate without electro elution. .. Dephosphorylation was achieved using shrimp alkaline phosphatase (Affymetrix Inc., USB) and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB).

    Ethanol Precipitation:

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: The substrates were prepared by annealing the 32 P-labeled oligonucleotide with a two-fold excess of the unlabeled oligonucleotide in a PNK buffer (New England Biolabs). .. The pUC19 plasmid was digested by HindIII-HF restriction enzyme and purified by phenol-chloroform extraction and ethanol precipitation.

    Immunoprecipitation:

    Article Title: C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1
    Article Snippet: C6 pyridinium ceramide treatment of HeLa nuclear extracts Ten micrograms of HeLa nuclear extract (NE) was incubated with 4 µCi [γ32 P] ATP for 30 min at 37°C in 1× PNK buffer (New England Bio Labs) plus or 10 µM C6 pyridinium ceramide in a 50 µl final volume. .. Radiolabeled-NE proteins were then subjected to immunoprecipitation overnight with 2 µg of indicated antibodies, 10 µl proteins A/G beads (Santa Cruz) and 200 µl RIPA-rescue buffer containing phosphatase and protease inhibitors.

    Cross-linking Immunoprecipitation:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Paragraph title: Crosslinking Immunoprecipitation and deep sequencing (CLIP-seq) in C. elegans ... The beads were washed three times with PNK Buffer and incubated in 80 μl PNK Buffer (NEB) with 40 units of T4 PNK enzyme (NEB) in the presence of 32 P-γ-ATP (1 mCi).

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    New England Biolabs t4 polynucleotide kinase
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