m0212  (New England Biolabs)


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    Structured Review

    New England Biolabs m0212
    M0212, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    m0212 - by Bioz Stars, 2021-07
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    New England Biolabs klenow fragment
    <t>DNA</t> synthesis activity of minimal reconstituted T4 replisomes across a minifork containing a random sequence. The minifork was prepared with the plasmid p-Empty and thus contains a random sequence to be replicated. gp43, gp41, and gp59 are the DNA polymerase, the helicase and the helicase loader of bacteriophage T4, respectively. (A) Gp43 was incubated with the minifork, alone (lane 2), with gp41 (lanes 3–8) or with gp41 and gp59 (lanes 9–15). The reaction was quenched at various times and the samples were loaded on a denaturing sequencing gel. (a) corresponds to the radiolabelled p821 primer. (b) corresponds to radiolabelled p821 extended by 15 nts up to the base of the 5′ ss tail of the lagging strand template. (c) corresponds to the radiolabelled p821 extended up to the end of the leading strand template after strand displacement DNA synthesis. The (a), (b), and (c) DNAs are also shown in the context of the minifork on the right side of the figure. Major transient intermediate products are indicated by backward arrows. The four sequencing reactions (A, T, C, and G) of the leading strand template of the minifork are shown on the left side of the figure. (B) The minifork was incubated with <t>Klenow</t> fragment alone (lanes 2 and 5), together with gp41 (lanes 3 and 6) or with gp41 and gp59 (lanes 4 and 7) for 20 minutes. The reaction products were resolved on a denaturing sequencing gel. Two amounts of Klenow fragment were tested (1 mU/ μ L, lanes 2–4; 10 mU/ μ L, lanes 5–7). (a) and (c) are as in 3A.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA synthesis activity of minimal reconstituted T4 replisomes across a minifork containing a random sequence. The minifork was prepared with the plasmid p-Empty and thus contains a random sequence to be replicated. gp43, gp41, and gp59 are the DNA polymerase, the helicase and the helicase loader of bacteriophage T4, respectively. (A) Gp43 was incubated with the minifork, alone (lane 2), with gp41 (lanes 3–8) or with gp41 and gp59 (lanes 9–15). The reaction was quenched at various times and the samples were loaded on a denaturing sequencing gel. (a) corresponds to the radiolabelled p821 primer. (b) corresponds to radiolabelled p821 extended by 15 nts up to the base of the 5′ ss tail of the lagging strand template. (c) corresponds to the radiolabelled p821 extended up to the end of the leading strand template after strand displacement DNA synthesis. The (a), (b), and (c) DNAs are also shown in the context of the minifork on the right side of the figure. Major transient intermediate products are indicated by backward arrows. The four sequencing reactions (A, T, C, and G) of the leading strand template of the minifork are shown on the left side of the figure. (B) The minifork was incubated with Klenow fragment alone (lanes 2 and 5), together with gp41 (lanes 3 and 6) or with gp41 and gp59 (lanes 4 and 7) for 20 minutes. The reaction products were resolved on a denaturing sequencing gel. Two amounts of Klenow fragment were tested (1 mU/ μ L, lanes 2–4; 10 mU/ μ L, lanes 5–7). (a) and (c) are as in 3A.

    Journal: Molecular Biology International

    Article Title: 5?CAG and 5?CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

    doi: 10.4061/2011/213824

    Figure Lengend Snippet: DNA synthesis activity of minimal reconstituted T4 replisomes across a minifork containing a random sequence. The minifork was prepared with the plasmid p-Empty and thus contains a random sequence to be replicated. gp43, gp41, and gp59 are the DNA polymerase, the helicase and the helicase loader of bacteriophage T4, respectively. (A) Gp43 was incubated with the minifork, alone (lane 2), with gp41 (lanes 3–8) or with gp41 and gp59 (lanes 9–15). The reaction was quenched at various times and the samples were loaded on a denaturing sequencing gel. (a) corresponds to the radiolabelled p821 primer. (b) corresponds to radiolabelled p821 extended by 15 nts up to the base of the 5′ ss tail of the lagging strand template. (c) corresponds to the radiolabelled p821 extended up to the end of the leading strand template after strand displacement DNA synthesis. The (a), (b), and (c) DNAs are also shown in the context of the minifork on the right side of the figure. Major transient intermediate products are indicated by backward arrows. The four sequencing reactions (A, T, C, and G) of the leading strand template of the minifork are shown on the left side of the figure. (B) The minifork was incubated with Klenow fragment alone (lanes 2 and 5), together with gp41 (lanes 3 and 6) or with gp41 and gp59 (lanes 4 and 7) for 20 minutes. The reaction products were resolved on a denaturing sequencing gel. Two amounts of Klenow fragment were tested (1 mU/ μ L, lanes 2–4; 10 mU/ μ L, lanes 5–7). (a) and (c) are as in 3A.

    Article Snippet: Similarly, the DNA synthesis pattern was examined in the presence of Klenow fragment.

    Techniques: DNA Synthesis, Activity Assay, Sequencing, Plasmid Preparation, Incubation

    DNA ligase I depletion in S. cerevisiae leads to the accumulation of Okazaki fragments sized similarly to the nucleosome repeat a , Transcriptional repression of DNA ligase I ( CDC9 ) results in the accumulation of nicked DNA. Cells carrying a doxycycline-repressible allele of the CDC9 gene were treated with doxycycline (Dox) for the indicated time. Purified genomic DNA was labelled using exonuclease-deficient Klenow fragment and α- 32 P dCTP and separated in a denaturing agarose gel. nt, nucleotides. b , The size of labelled Okazaki fragments mirrors the nucleosome repeat. Okazaki fragments (lane 2) were labelled as in Fig. 1a; nucleosomes were prepared from wild-type (lane 3) or repressible CDC9 strains (lanes 4 and 5) by in vivo Micrococcal Nuclease (MNase) digestion. The chromatin digestion patterns in lanes 4 and 5 indicate that CDC9 repression does not alter global chromatin structure. c , Okazaki fragments accumulate during S phase. Cells were arrested in G1 using α-factor, during which time CDC9 transcription was inhibited by the addition of doxycycline, and degradation of the protein stimulated by activation of the degron system using galactose and 37 °C. Okazaki fragments appear upon release of the culture into S phase (lanes 3 and 4). d, Okazaki fragments are bordered by ligatable nicks. Purified DNA was treated (lanes 2 and 4) or mock-treated (lanes 1 and 3) with the indicated ligase, and then labelled as in Fig. 1a. The inability to label fragments after ligase treatment confirms that labelled Okazaki fragments are flanked by ligatable nicks.

    Journal: Nature

    Article Title: Intrinsic coupling of lagging-strand synthesis to chromatin assembly

    doi: 10.1038/nature10895

    Figure Lengend Snippet: DNA ligase I depletion in S. cerevisiae leads to the accumulation of Okazaki fragments sized similarly to the nucleosome repeat a , Transcriptional repression of DNA ligase I ( CDC9 ) results in the accumulation of nicked DNA. Cells carrying a doxycycline-repressible allele of the CDC9 gene were treated with doxycycline (Dox) for the indicated time. Purified genomic DNA was labelled using exonuclease-deficient Klenow fragment and α- 32 P dCTP and separated in a denaturing agarose gel. nt, nucleotides. b , The size of labelled Okazaki fragments mirrors the nucleosome repeat. Okazaki fragments (lane 2) were labelled as in Fig. 1a; nucleosomes were prepared from wild-type (lane 3) or repressible CDC9 strains (lanes 4 and 5) by in vivo Micrococcal Nuclease (MNase) digestion. The chromatin digestion patterns in lanes 4 and 5 indicate that CDC9 repression does not alter global chromatin structure. c , Okazaki fragments accumulate during S phase. Cells were arrested in G1 using α-factor, during which time CDC9 transcription was inhibited by the addition of doxycycline, and degradation of the protein stimulated by activation of the degron system using galactose and 37 °C. Okazaki fragments appear upon release of the culture into S phase (lanes 3 and 4). d, Okazaki fragments are bordered by ligatable nicks. Purified DNA was treated (lanes 2 and 4) or mock-treated (lanes 1 and 3) with the indicated ligase, and then labelled as in Fig. 1a. The inability to label fragments after ligase treatment confirms that labelled Okazaki fragments are flanked by ligatable nicks.

    Article Snippet: Two microlitres of DNA (corresponding to the genomic DNA content of 2 ml cultured cells) was used in 20 μl labelling reactions containing 5 U Klenow (exo-)polymerase (NEB) and α-dCTP (Perkin Elmer) at a final concentration of 33 nM.

    Techniques: Purification, Agarose Gel Electrophoresis, In Vivo, Activation Assay

    Assay schemes. a Strategy for total genomic methylation. Genomic DNA is restriction enzyme ( RE ) digested, enzymatically biotinylated via a fill-in reaction with Klenow polymerase and biotin-dNTPs. MBD magnetic beads are then used to select for methylated DNA. Colorimetric evaluation is mediated by SA-HRP which recognizes the biotin on enriched methylated DNA. b Strategy for gene-specific methylation. Genomic DNA is RE digested and methylated DNA is selected via MBD enrichment. Gene-specific isothermal amplification is then performed with biotin-dNTP to generate biotin-DNA polymers which are in turn selected for with SA magnetic beads and SA-HRP for colorimetric evaluation

    Journal: Clinical Epigenetics

    Article Title: Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs

    doi: 10.1186/s13148-015-0100-6

    Figure Lengend Snippet: Assay schemes. a Strategy for total genomic methylation. Genomic DNA is restriction enzyme ( RE ) digested, enzymatically biotinylated via a fill-in reaction with Klenow polymerase and biotin-dNTPs. MBD magnetic beads are then used to select for methylated DNA. Colorimetric evaluation is mediated by SA-HRP which recognizes the biotin on enriched methylated DNA. b Strategy for gene-specific methylation. Genomic DNA is RE digested and methylated DNA is selected via MBD enrichment. Gene-specific isothermal amplification is then performed with biotin-dNTP to generate biotin-DNA polymers which are in turn selected for with SA magnetic beads and SA-HRP for colorimetric evaluation

    Article Snippet: After 30 min, the reaction was supplemented to a final volume of 25 μL with 5 units of Klenow fragment (3′→5′ exo-) DNA polymerase (NEB, USA) and 5 μM of biotin-14-dUTP, dATP, dGTP and dCTP and incubated at 37 °C for another 30 min to fill in the overhangs and biotinylate the fragmented DNA.

    Techniques: Methylation, Magnetic Beads, Amplification

    UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Article Snippet: The absence of any products longer than ~100 nucleotides in assays containing UL30 is not due to either the lower processivity of UL30 relative to UL30-UL42 since UL30 and Klenow Fragment have similar processivity ( ) and Klenow Fragment generates long products.

    Techniques: Agarose Gel Electrophoresis

    Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Article Snippet: The absence of any products longer than ~100 nucleotides in assays containing UL30 is not due to either the lower processivity of UL30 relative to UL30-UL42 since UL30 and Klenow Fragment have similar processivity ( ) and Klenow Fragment generates long products.

    Techniques: Agarose Gel Electrophoresis