mmei  (New England Biolabs)


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  • 93
    Name:
    MmeI
    Description:
    MmeI 500 units
    Catalog Number:
    r0637l
    Price:
    282
    Size:
    500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs mmei
    MmeI
    MmeI 500 units
    https://www.bioz.com/result/mmei/product/New England Biolabs
    Average 93 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    mmei - by Bioz Stars, 2020-05
    93/100 stars

    Images

    1) Product Images from "Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities"

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002442

    MmeI/DNA/Sinefungin ternary complex. (A) The MmeI helicase spacer (residues 156–300), methyltransferase (residues 301–620), DNA target recognition domain (TRD; residues 621–825), and C-terminal helical region (residues 826–919) are shown in purple, green, cyan, and orange, respectively. DNA is in yellow. The flipped-out adenine (orange) and the bound Sinefungin (red) are labeled. (B) Crystallized DNA and Sinefungin with accompanying 2Fo-Fc map contoured at 1.5 σ. The recognition sequence of TCCGAC is labeled along with the complementary strand.
    Figure Legend Snippet: MmeI/DNA/Sinefungin ternary complex. (A) The MmeI helicase spacer (residues 156–300), methyltransferase (residues 301–620), DNA target recognition domain (TRD; residues 621–825), and C-terminal helical region (residues 826–919) are shown in purple, green, cyan, and orange, respectively. DNA is in yellow. The flipped-out adenine (orange) and the bound Sinefungin (red) are labeled. (B) Crystallized DNA and Sinefungin with accompanying 2Fo-Fc map contoured at 1.5 σ. The recognition sequence of TCCGAC is labeled along with the complementary strand.

    Techniques Used: Labeling, Sequencing

    Change in specificity at position 2. (A) Restriction fragment digestion patterns of lambda, PhiX174, and pBR322 DNAs with wt = wild type MmeI, which cuts at TCCRAC20/18; A = MmeI Lys 645 Met mutant, which cuts at TACRAC20/18; R = MmeI Tyr 642 Lys, Lys 645 Met double mutant, which cuts at TRCRAC20/18; M = size standard, lambda-HindIII digest plus PhiX174-HaeIII digest. (B) Cut site determination for MmeI K 645 M mutant showing cutting at TACRAC20/18. Run-off Sanger sequencing of pUC19 DNA (TACRAC site at 376 to 381), priming from both sides (5' and 3') of the TACRAC recognition site and point of DNA cleavage. (C) Cut site determination for MmeI Y 642 K, K 645 M double mutant showing cutting at both TACRAC20/18 (top panel, pUC19 site at 376 to 381) and TGCRAC (bottom panel, pUC19 site at 1842 to 1847). Run-off Sanger sequencing of the cleaved pUC19 DNA, showing priming from 5' to the TACRAC or TGCRAC recognition site (bottom strand cleavage shown).
    Figure Legend Snippet: Change in specificity at position 2. (A) Restriction fragment digestion patterns of lambda, PhiX174, and pBR322 DNAs with wt = wild type MmeI, which cuts at TCCRAC20/18; A = MmeI Lys 645 Met mutant, which cuts at TACRAC20/18; R = MmeI Tyr 642 Lys, Lys 645 Met double mutant, which cuts at TRCRAC20/18; M = size standard, lambda-HindIII digest plus PhiX174-HaeIII digest. (B) Cut site determination for MmeI K 645 M mutant showing cutting at TACRAC20/18. Run-off Sanger sequencing of pUC19 DNA (TACRAC site at 376 to 381), priming from both sides (5' and 3') of the TACRAC recognition site and point of DNA cleavage. (C) Cut site determination for MmeI Y 642 K, K 645 M double mutant showing cutting at both TACRAC20/18 (top panel, pUC19 site at 376 to 381) and TGCRAC (bottom panel, pUC19 site at 1842 to 1847). Run-off Sanger sequencing of the cleaved pUC19 DNA, showing priming from 5' to the TACRAC or TGCRAC recognition site (bottom strand cleavage shown).

    Techniques Used: Mutagenesis, Sequencing

    Structural comparison between MmeI and BpuSI. (A) MmeI (red) bound to DNA (yellow) superimposed on apo BpuSI (cyan). The helicase connector, methyltransferase, and DNA target recognition domain (TRD) labels correspond to both structures, while the endonuclease domain is only visible in the BpuSI structure. A comparison of the two structures reveals an ~38° rotation in the TRD, which clamps down on the DNA to make specific contacts. The TRD as a whole shifts by ~27 Å between the two structures. (B) A 90° rotation of the view in (a) to show the relative position of the endonuclease domain.
    Figure Legend Snippet: Structural comparison between MmeI and BpuSI. (A) MmeI (red) bound to DNA (yellow) superimposed on apo BpuSI (cyan). The helicase connector, methyltransferase, and DNA target recognition domain (TRD) labels correspond to both structures, while the endonuclease domain is only visible in the BpuSI structure. A comparison of the two structures reveals an ~38° rotation in the TRD, which clamps down on the DNA to make specific contacts. The TRD as a whole shifts by ~27 Å between the two structures. (B) A 90° rotation of the view in (a) to show the relative position of the endonuclease domain.

    Techniques Used:

    Related Articles

    DNA Cleavage Assay:

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
    Article Snippet: .. DNA Cleavage Assay Endonuclease activity was assayed by incubating various amounts of MmeI (wt or mutant) enzyme for 30 min at 37°C in NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) supplemented with AdoMet at 80 μM, containing 1 μg substrate DNA per 50 μl. .. Reactions were terminated by the addition of loading dye (NEB B7024) and reaction products were analyzed by gel electrophoresis in 1% LE agarose gels.

    Ligation:

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
    Article Snippet: .. MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: .. Ligation to Linker B Linker B was purchased as two separate oligonucleotides (5'P-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCGGTGGTCGCCGTATCATT-OH3', 5'OH-TCATCTTTCCCTACACGACGCTCTTCCGATCTNN-OH3') and hybridized using the same procedure as described for Linker A. Mme I products were ligated to Linker B using the following 50 μl reaction mix: 10.0 μl Mme I product, 1.0 μl of 0.05 mM Linker B, 5.0 μl 10× ligase buffer, 3.0 μl T4 DNA ligase (2,000 U/μl; NEB #M0202), 31.0 μl dH2 O. Ligations were performed overnight using a PCR machine. ..

    Mutagenesis:

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
    Article Snippet: .. DNA Cleavage Assay Endonuclease activity was assayed by incubating various amounts of MmeI (wt or mutant) enzyme for 30 min at 37°C in NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) supplemented with AdoMet at 80 μM, containing 1 μg substrate DNA per 50 μl. .. Reactions were terminated by the addition of loading dye (NEB B7024) and reaction products were analyzed by gel electrophoresis in 1% LE agarose gels.

    Isolation:

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
    Article Snippet: .. MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

    Purification:

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
    Article Snippet: .. Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification. .. Prepare Illumina P5 and P7 adapters by annealing overhang NN-nucleotides P5: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3′ and 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′; P7: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ and 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNN-3′, respectively.

    Incubation:

    Article Title: Targeted and genome-scale methylomics reveals gene body signatures in human cell lines
    Article Snippet: .. This was incubated at 50°C for 20 min, then 85°C for 20 min. Ethanol precipitation was performed again, and the pellet was resuspended to 50ul with a reaction mixture containing 2 units Mme I (NEB), 50 µM SAM and 1× NEBuffer 4. .. This was incubated at 37°C for 2 hours, then 80°C for 20 min. To this was added 1.66 µl of 10 µM adapter B, 6 µl 10mM ATP, and 3 µl T4 DNA ligase, and the mixture was incubated at 16°C for 4 hours, then 65°C for 15 min.

    other:

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: Mme I digestion Linker A-ligated molecules were subjected to Mme I digestion using the following 200 μl reaction mix: 20 μl Linker A-ligated product, 20.0 μl 10× NEB 4, 0.3 μl 32 mM S-adenosyl methionine, 2.0 μl Mme I (2 U/μl; NEB R0637), 157.7 μl dH2 O.

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: MATERIALS AND METHODS MmeI, AdoMet, all restriction endonucleases, T4 DNA Ligase, Phusion DNA polymerase, DNA size standards and competent cells were obtained from New England Biolabs (Ipswich, MA).

    Activity Assay:

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
    Article Snippet: .. DNA Cleavage Assay Endonuclease activity was assayed by incubating various amounts of MmeI (wt or mutant) enzyme for 30 min at 37°C in NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) supplemented with AdoMet at 80 μM, containing 1 μg substrate DNA per 50 μl. .. Reactions were terminated by the addition of loading dye (NEB B7024) and reaction products were analyzed by gel electrophoresis in 1% LE agarose gels.

    Ethanol Precipitation:

    Article Title: Targeted and genome-scale methylomics reveals gene body signatures in human cell lines
    Article Snippet: .. This was incubated at 50°C for 20 min, then 85°C for 20 min. Ethanol precipitation was performed again, and the pellet was resuspended to 50ul with a reaction mixture containing 2 units Mme I (NEB), 50 µM SAM and 1× NEBuffer 4. .. This was incubated at 37°C for 2 hours, then 80°C for 20 min. To this was added 1.66 µl of 10 µM adapter B, 6 µl 10mM ATP, and 3 µl T4 DNA ligase, and the mixture was incubated at 16°C for 4 hours, then 65°C for 15 min.

    Polymerase Chain Reaction:

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: .. Ligation to Linker B Linker B was purchased as two separate oligonucleotides (5'P-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCGGTGGTCGCCGTATCATT-OH3', 5'OH-TCATCTTTCCCTACACGACGCTCTTCCGATCTNN-OH3') and hybridized using the same procedure as described for Linker A. Mme I products were ligated to Linker B using the following 50 μl reaction mix: 10.0 μl Mme I product, 1.0 μl of 0.05 mM Linker B, 5.0 μl 10× ligase buffer, 3.0 μl T4 DNA ligase (2,000 U/μl; NEB #M0202), 31.0 μl dH2 O. Ligations were performed overnight using a PCR machine. ..

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
    Article Snippet: .. Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification. .. Prepare Illumina P5 and P7 adapters by annealing overhang NN-nucleotides P5: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3′ and 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′; P7: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ and 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNN-3′, respectively.

    Gel Extraction:

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
    Article Snippet: .. MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Plasmid Preparation:

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Gel Purification:

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
    Article Snippet: .. Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification. .. Prepare Illumina P5 and P7 adapters by annealing overhang NN-nucleotides P5: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3′ and 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′; P7: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ and 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNN-3′, respectively.

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    New England Biolabs gel loading dye purple 6x
    Gel Loading Dye Purple 6x, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95/100 stars
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