mmei site  (New England Biolabs)


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    Structured Review

    New England Biolabs mmei site
    <t>MmeI</t> cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled <t>pUC19</t> DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.
    Mmei Site, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mmei site/product/New England Biolabs
    Average 87 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mmei site - by Bioz Stars, 2020-04
    87/100 stars

    Images

    1) Product Images from "MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection"

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn711

    MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.
    Figure Legend Snippet: MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.

    Techniques Used: Plasmid Preparation, Serial Dilution

    ( A ) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N 773 D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. ( B ) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. ( C ) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.
    Figure Legend Snippet: ( A ) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N 773 D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. ( B ) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. ( C ) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.

    Techniques Used: In Vivo, Modification, Purification, Plasmid Preparation, Construct, Expressing, Derivative Assay, Mutagenesis, In Vitro

    Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC GACTC -3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG GATC -3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.
    Figure Legend Snippet: Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC GACTC -3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG GATC -3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.

    Techniques Used: In Vitro, Modification, Polymerase Chain Reaction

    Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).
    Figure Legend Snippet: Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).

    Techniques Used: Modification, In Vitro, In Vivo, Methylation, Plasmid Preparation

    Related Articles

    In Vitro:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: .. The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

    Positive Control:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The MmeI reaction was split and 1 μg unmodified pMMH1 DNA was added to one aliquot as a positive control.

    Methylation:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: .. The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

    Mutagenesis:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: .. The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

    Introduce:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: .. The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

    Purification:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

    Activity Assay:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: Paragraph title: MmeI methylation effect on MmeI endonuclease activity ... The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7.

    Modification:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

    Plasmid Preparation:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: .. The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7. .. The HinfI site in pUC19 at position 427 was removed by the mutagenesis. pMMH1 was grown in a dam methylase-proficient host, ER2683, or in a dam methylase-deficient host, ER2925 (to avoid 5′-GATC-3′ adenine methylation), purified and sequenced to confirm the mutagenic alteration. pMMH1 DNA from the dam minus host was modified in vitro by MmeI, purified over a spin column and digested with MmeI, MboI and HinfI.

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    New England Biolabs mmei site
    <t>MmeI</t> cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled <t>pUC19</t> DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.
    Mmei Site, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mmei site/product/New England Biolabs
    Average 87 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mmei site - by Bioz Stars, 2020-04
    87/100 stars
      Buy from Supplier

    85
    New England Biolabs mmei restriction enzyme recognition site
    <t>MmeI</t> cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled <t>pUC19</t> DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.
    Mmei Restriction Enzyme Recognition Site, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mmei restriction enzyme recognition site/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mmei restriction enzyme recognition site - by Bioz Stars, 2020-04
    85/100 stars
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    MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.

    Journal: Nucleic Acids Research

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    doi: 10.1093/nar/gkn711

    Figure Lengend Snippet: MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.

    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7.

    Techniques: Plasmid Preparation, Serial Dilution

    ( A ) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N 773 D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. ( B ) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. ( C ) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.

    Journal: Nucleic Acids Research

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    doi: 10.1093/nar/gkn711

    Figure Lengend Snippet: ( A ) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N 773 D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. ( B ) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. ( C ) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.

    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7.

    Techniques: In Vivo, Modification, Purification, Plasmid Preparation, Construct, Expressing, Derivative Assay, Mutagenesis, In Vitro

    Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC GACTC -3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG GATC -3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.

    Journal: Nucleic Acids Research

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    doi: 10.1093/nar/gkn711

    Figure Lengend Snippet: Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC GACTC -3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG GATC -3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.

    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7.

    Techniques: In Vitro, Modification, Polymerase Chain Reaction

    Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).

    Journal: Nucleic Acids Research

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    doi: 10.1093/nar/gkn711

    Figure Lengend Snippet: Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).

    Article Snippet: The effect of in vitro MmeI methylation was tested using a plasmid substrate, pMMH1, designed to introduce an MmeI site in the pUC19 polylinker region that overlaps a 5′ MboI site (GATC) and a 3′ HinfI site (GANTC): 5′-… cccgggGATCCGACTCcggtcgac …-3′. pMMH1 was formed from pUC19 using the Phusion Site-Directed Mutagenesis Kit (NEB) with primers 6 and 7.

    Techniques: Modification, In Vitro, In Vivo, Methylation, Plasmid Preparation