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    Name:
    MmeI
    Description:
    MmeI 500 units
    Catalog Number:
    r0637l
    Price:
    282
    Size:
    500 units
    Category:
    Restriction Enzymes
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    New England Biolabs mmei new england biolabs
    MmeI
    MmeI 500 units
    https://www.bioz.com/result/mmei new england biolabs/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mmei new england biolabs - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Paired-end Tag:

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs). .. Tags were self-ligated to form ditags, which were then further ligated to form concatemers and cloned into pZero (Invitrogen).

    Article Title: 5? Long serial analysis of gene expression (LongSAGE) and 3? LongSAGE for transcriptome characterization and genome annotation
    Article Snippet: Formation of 5′LS ditags. .. After size fractionation, the selected cDNA was immobilized and digested with Mme I (New England Biolabs, Beverly, MA) to release the 5′-terminal tags.

    Centrifugation:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: At each time point, 10 ml aliquots were collected to harvest cells by centrifugation for subsequent gDNA isolation. .. Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction.

    Amplification:

    Article Title: Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation
    Article Snippet: PCR amplification of the linked cDNAs was performed with primers that anneal in the GAL4 DBD and GAL4 AD cDNA. .. It was then digested by MmeI New England Biolabs (NEB) and purified by 6% PAGE and the excised band was eluted into TE.

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: .. Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs). .. After treatment with Klenow Enzyme (New England Biolabs), indexed libraries were prepared with a Multiplexing Sample Preparation Oligonucleotide Kit (Illumina) according to the user's manual.

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA). .. The ligated products were amplified and the resulting product was sequenced using an Illumina HiSeq™ 2000 system, then the data process using PAIRFINDER (version 2.0) Hao et al. [ ].

    Article Title: 5? Long serial analysis of gene expression (LongSAGE) and 3? LongSAGE for transcriptome characterization and genome annotation
    Article Snippet: After size fractionation, the selected cDNA was immobilized and digested with Mme I (New England Biolabs, Beverly, MA) to release the 5′-terminal tags. .. The released tags were then pooled and ligated to form 5′LS ditags that were amplified by PCR.

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: Primary PCR amplification of the library was conducted on a qPCR thermal cycler with a 5′ PCR primer (AAG CAG TGG TAT CAA CGC AGA GT ) and the 3′ synthesis primer. .. Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below).

    Clone Assay:

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs). .. Tags were self-ligated to form ditags, which were then further ligated to form concatemers and cloned into pZero (Invitrogen).

    Article Title: 5? Long serial analysis of gene expression (LongSAGE) and 3? LongSAGE for transcriptome characterization and genome annotation
    Article Snippet: After size fractionation, the selected cDNA was immobilized and digested with Mme I (New England Biolabs, Beverly, MA) to release the 5′-terminal tags. .. Concatenation and cloning of 5′LS ditags.

    Construct:

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: To predict the potential target genes of the miRNA, three degradome libraries were constructed according to the methods of Addo-Quaye and German [ , ]. .. Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA).

    Article Title: Genome of Paulownia (Paulownia fortunei) illuminates the related transcripts, miRNA and proteins for salt resistance
    Article Snippet: Degradome sequencing and Identification of miRNA targets Four degradome libraries corresponding to PF2, PF4, PF2S and PF4S were constructed as described previously . .. RNAs with poly (A) in the 3′adapter and a Mme I (NEB, Ipswich, MA, USA) recognition site in the 5′ adapter were isolated.

    Article Title: Targeted and genome-scale methylomics reveals gene body signatures in human cell lines
    Article Snippet: To construct the MSCC Hpa II library, 2 µg of PGP 1 lymphocyte gDNA were assembled into a 100 µl reaction with 20 units Hpa II (NEB) in 1× NEBuffer 1, incubated at 37°C 2 hours, then 65°C 20 min. To this was added 1.66 µl of 10 µM adapter A, 12 µl 10 mM ATP, and 120 units T4 DNA ligase (NEB). .. 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.

    Electrophoresis:

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: Double-stranded cDNA was purified by QIAquick PCR purification kit (Qiagen) and checked by electrophoresis on a 1.5% agarose gel. .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    cDNA Library Assay:

    Article Title: Genome of Paulownia (Paulownia fortunei) illuminates the related transcripts, miRNA and proteins for salt resistance
    Article Snippet: RNAs with poly (A) in the 3′adapter and a Mme I (NEB, Ipswich, MA, USA) recognition site in the 5′ adapter were isolated. .. The final cDNA library was purified and sequenced using an Illumina HiSeq™ 2000 system (Beijing Genomics Institute, Shenzhen, China).

    Incubation:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: 10 ml of the resulting T1 culture was inoculated in 250 ml THY containing Km and incubated overnight at 37°C a second (T2 ) and third (T3 ) time. .. Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction.

    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.

    Expressing:

    Article Title: Genetic dissection of blood lipid traits by integrating genome-wide association study and gene expression profiling in a porcine model
    Article Snippet: QTT analysis and eQTL mapping Digital gene expression (DGE) analyses of genome-wide transcripts were performed on 497 F2 liver samples as described previously [ ]. .. Using the mRNA attached to the bead as a template, double-stranded cDNA was synthesized with oligo-d (T) primers, and then digested with restriction enzymes Nla III and Mme I (New England Biolabs, UK).

    Genome Wide:

    Article Title: Genetic dissection of blood lipid traits by integrating genome-wide association study and gene expression profiling in a porcine model
    Article Snippet: QTT analysis and eQTL mapping Digital gene expression (DGE) analyses of genome-wide transcripts were performed on 497 F2 liver samples as described previously [ ]. .. Using the mRNA attached to the bead as a template, double-stranded cDNA was synthesized with oligo-d (T) primers, and then digested with restriction enzymes Nla III and Mme I (New England Biolabs, UK).

    Modification:

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below). .. The second library was prepared like the first, except that the 3′ adapter was modified to include a MmeI site (PD243Mme-30TC - ATT CTA GAG CGC ACC TTG GCC TCC GAC TTT TCT TTT CTT TTT TTT TCT TTT TTT TTT VN).

    Ligation:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. .. The ligation mixture was then used as a template for a 20-cycle PCR with the primers oKrmit-Tnseq2 and oAdapterPCR , resulting in the production of 176-bp Krmit insertion tags ( ) that were purified from a 2% agarose gel.

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. Ligation products were used a templates in replicate PCR reactions with high-fidelity polymerase (KAPA Biosystems) and the LIB_PCR_5 and LIB_PCR_3 primers [ ].

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Following ligation of a pair of top-Phops-oligo-17bp/T7Mme I-18bp adaptors, fragments between 250 and 650 bp were isolated from agarose gel. .. Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs).

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. Ligation products were used a templates in replicate PCR reactions with high-fidelity polymerase (KAPA Biosystems) and the LIB_PCR_5 and LIB_PCR_3 primers [ ].

    Library Screening:

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Paragraph title: Transposon library screening ... Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size.

    SYBR Green Assay:

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: A control reaction was spiked with 1 x SYBR green (Invitrogen S7563) to monitor for overcycling. .. Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below).

    Synthesized:

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: Second strand cDNA was then synthesized by low cycle primer extension using Advantage® 2 polymerase (Clontech). .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    Article Title: Genetic dissection of blood lipid traits by integrating genome-wide association study and gene expression profiling in a porcine model
    Article Snippet: .. Using the mRNA attached to the bead as a template, double-stranded cDNA was synthesized with oligo-d (T) primers, and then digested with restriction enzymes Nla III and Mme I (New England Biolabs, UK). .. The digested-cDNA was ligated to Illumina specific adapters 1 and 2.

    Article Title: Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1 [w]
    Article Snippet: The linker oligonucleotides, i.e. linker 1 (linker 1A [5′-TTTGGATTTGCTGGTGCAGTACAACT AGGCTTAATATCCGACATG-3′] and linker 1B [5′-TCGGATATTAAGCCTAGTTGT ACTGCACCAGCAAATCC-C7 amino-modified-3′]) and linker 2 (linker 2A [5′-TTTCT GCTCGAATTCAAGCTTCTAACGATGTACGTCCGACATG-3′ and linker 2B 5′-TC GGACGTACATCG TTAGAAGCTTGAATTCGAGCAG-C7 amino-modified-3′]) were synthesized and purified on a polyacrylamide gel (Integrated DNA Technologies Inc., Coralville, IA) as reported by Saha et al. ( ). .. After the beads were washed thoroughly, pools A and B were treated with 20 units of Mme I (37°C, 3 h) (New England Biolabs, Inc., Beverly, MA).

    Generated:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. .. Eight different Mme I adapters (Adapter-501 to Adapter-508) were generated by annealing oligonucleotide pairs ( ) containing Illumina barcode sequences (501 to 508) to allow sample multiplexing during massively parallel sequencing.

    Inhibition:

    Article Title: 5? Long serial analysis of gene expression (LongSAGE) and 3? LongSAGE for transcriptome characterization and genome annotation
    Article Snippet: As in the standard SAGE procedure, to avoid PCR inhibition, the cDNA pool was first divided into two aliquots, each of which was then ligated to an adapter differing in the 5′ PCR primer annealing region. .. After size fractionation, the selected cDNA was immobilized and digested with Mme I (New England Biolabs, Beverly, MA) to release the 5′-terminal tags.

    Sequencing:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. .. Eight different Mme I adapters (Adapter-501 to Adapter-508) were generated by annealing oligonucleotide pairs ( ) containing Illumina barcode sequences (501 to 508) to allow sample multiplexing during massively parallel sequencing.

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. Pooled replicate reactions were sequenced by Illumina HiSeq 2500 at the Harvard Biopolymers Sequencing Facility.

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Paragraph title: bPPI-seq library preparation and sequencing ... Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs).

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA). .. The small RNA and degradome sequencing data used in this publication have been deposited in the NIH Short Read Archive database (http:// http://www.ncbi.nlm.nih.gov/sra ) and the study accession number are SRP060300 and SRP060876.

    Article Title: Genome of Paulownia (Paulownia fortunei) illuminates the related transcripts, miRNA and proteins for salt resistance
    Article Snippet: Paragraph title: Degradome sequencing and Identification of miRNA targets ... RNAs with poly (A) in the 3′adapter and a Mme I (NEB, Ipswich, MA, USA) recognition site in the 5′ adapter were isolated.

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs). .. Clones were sequenced by using Big Dye terminators (Applied Biosystems) and analyzed with a 384-capillary automated sequencing apparatus (Spectrumedix, State College, PA) or a 96-capillary ABI3700 instrument at Agencourt Biosciences (Beverly, MA).

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. Pooled replicate reactions were sequenced by Illumina HiSeq 2500 at the Harvard Biopolymers Sequencing Facility.

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: Paragraph title: 454 sequencing ... Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below).

    Sonication:

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: The cDNAs were amplified by a GFPC-specific (VNC primer) and T7 primers, and fragmented to 200 to 500 bp by sonication. .. Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs).

    Binding Assay:

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: DNA fragments containing biotinylated linkers were isolated by binding to streptavidin-coated magnetic beads (Dynal). .. Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs).

    Cellular Antioxidant Activity Assay:

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: Primary PCR amplification of the library was conducted on a qPCR thermal cycler with a 5′ PCR primer (AAG CAG TGG TAT CAA CGC AGA GT ) and the 3′ synthesis primer. .. Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below).

    DNA Extraction:

    Article Title: Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn‐Seq analysis
    Article Snippet: Paragraph title: DNA extraction and Tn‐Seq processing ... Four micrograms of DNA from each sample were used in a Mme I (New England Biolabs, Ipswich, MA, USA) digestion reaction according to the manufacturer's instructions at 37 °C overnight.

    RNA Sequencing Assay:

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: Paragraph title: Small RNA sequencing, identification of miRNAs and their target genes ... Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA).

    Magnetic Beads:

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: DNA fragments containing biotinylated linkers were isolated by binding to streptavidin-coated magnetic beads (Dynal). .. Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs).

    Article Title: Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1 [w]
    Article Snippet: About 50 ng of poly(A+ ) mRNA was bound to magnetic beads with oligo(dT)25 , and cDNA was synthesized directly on the oligo(dT) beads. cDNA was digested with Nla III and divided equally into two parts, pools A and B. .. After the beads were washed thoroughly, pools A and B were treated with 20 units of Mme I (37°C, 3 h) (New England Biolabs, Inc., Beverly, MA).

    Mutagenesis:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: .. Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. ..

    Isolation:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: .. Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. ..

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Following ligation of a pair of top-Phops-oligo-17bp/T7Mme I-18bp adaptors, fragments between 250 and 650 bp were isolated from agarose gel. .. Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs).

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: In brief, poly (A) RNA was isolated from the total RNA of each sample using an Oligotex mRNA mini kit (Qiagen, Shanghai, China). .. Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA).

    Article Title: Genome of Paulownia (Paulownia fortunei) illuminates the related transcripts, miRNA and proteins for salt resistance
    Article Snippet: .. RNAs with poly (A) in the 3′adapter and a Mme I (NEB, Ipswich, MA, USA) recognition site in the 5′ adapter were isolated. ..

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: DNA fragments containing biotinylated linkers were isolated by binding to streptavidin-coated magnetic beads (Dynal). .. Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs).

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: Ditag formation Total RNA was extracted by TRI® reagent (Molecular Research Center, Inc) and Poly(A)+ mRNA was furthered isolated using the PolyATract® mRNA isolation system (Promega). .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    Article Title: Genetic dissection of blood lipid traits by integrating genome-wide association study and gene expression profiling in a porcine model
    Article Snippet: In brief, mRNA was isolated from total RNA with the magnetic oligo (dT) beads (invitrogen, USA). .. Using the mRNA attached to the bead as a template, double-stranded cDNA was synthesized with oligo-d (T) primers, and then digested with restriction enzymes Nla III and Mme I (New England Biolabs, UK).

    Multiplexing:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. .. Eight different Mme I adapters (Adapter-501 to Adapter-508) were generated by annealing oligonucleotide pairs ( ) containing Illumina barcode sequences (501 to 508) to allow sample multiplexing during massively parallel sequencing.

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs). .. After treatment with Klenow Enzyme (New England Biolabs), indexed libraries were prepared with a Multiplexing Sample Preparation Oligonucleotide Kit (Illumina) according to the user's manual.

    Purification:

    Article Title: Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation
    Article Snippet: .. It was then digested by MmeI New England Biolabs (NEB) and purified by 6% PAGE and the excised band was eluted into TE. ..

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: .. Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. ..

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. These products were ligated to double-stranded barcoded DNA adaptors using T4 DNA ligase (New England Biolabs) and purified (Qiaquick PCR Purification Kit).

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Purified RNAs were reverse transcribed using PrimeScript Reverse Transcriptase (Takara) with a PolyT primer. .. Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs).

    Article Title: Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn‐Seq analysis
    Article Snippet: Four micrograms of DNA from each sample were used in a Mme I (New England Biolabs, Ipswich, MA, USA) digestion reaction according to the manufacturer's instructions at 37 °C overnight. .. These reactions were purified with a QIAquick PCR purification kit (Qiagen, Germantown, MD, USA) and eluted in 60 µL of 10 m m Tris‐HCl.

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: .. Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA). ..

    Article Title: Genome of Paulownia (Paulownia fortunei) illuminates the related transcripts, miRNA and proteins for salt resistance
    Article Snippet: RNAs with poly (A) in the 3′adapter and a Mme I (NEB, Ipswich, MA, USA) recognition site in the 5′ adapter were isolated. .. The final cDNA library was purified and sequenced using an Illumina HiSeq™ 2000 system (Beijing Genomics Institute, Shenzhen, China).

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. These products were ligated to double-stranded barcoded DNA adaptors using T4 DNA ligase (New England Biolabs) and purified (Qiaquick PCR Purification Kit).

    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. .. The mixture was run on a 6% non-denaturing TBE polyacrylamide gel (Invitrogen) and the target band at ~140 bp was purified.

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: Double-stranded cDNA was purified by QIAquick PCR purification kit (Qiagen) and checked by electrophoresis on a 1.5% agarose gel. .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: .. Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below). .. Products were size selected using Chromaspin TE-400 columns (Clontech#636076), blunted using NEB kit (NEB# E1201L), and quantified.

    Article Title: Genetic dissection of blood lipid traits by integrating genome-wide association study and gene expression profiling in a porcine model
    Article Snippet: Using the mRNA attached to the bead as a template, double-stranded cDNA was synthesized with oligo-d (T) primers, and then digested with restriction enzymes Nla III and Mme I (New England Biolabs, UK). .. After purification and denaturation, the single chain molecules of each cDNA library were loaded onto the flowcell and sequenced on a GA II sequencer (Illumina, USA).

    Article Title: Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1 [w]
    Article Snippet: The linker oligonucleotides, i.e. linker 1 (linker 1A [5′-TTTGGATTTGCTGGTGCAGTACAACT AGGCTTAATATCCGACATG-3′] and linker 1B [5′-TCGGATATTAAGCCTAGTTGT ACTGCACCAGCAAATCC-C7 amino-modified-3′]) and linker 2 (linker 2A [5′-TTTCT GCTCGAATTCAAGCTTCTAACGATGTACGTCCGACATG-3′ and linker 2B 5′-TC GGACGTACATCG TTAGAAGCTTGAATTCGAGCAG-C7 amino-modified-3′]) were synthesized and purified on a polyacrylamide gel (Integrated DNA Technologies Inc., Coralville, IA) as reported by Saha et al. ( ). .. After the beads were washed thoroughly, pools A and B were treated with 20 units of Mme I (37°C, 3 h) (New England Biolabs, Inc., Beverly, MA).

    Polymerase Chain Reaction:

    Article Title: Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation
    Article Snippet: PCR amplification of the linked cDNAs was performed with primers that anneal in the GAL4 DBD and GAL4 AD cDNA. .. It was then digested by MmeI New England Biolabs (NEB) and purified by 6% PAGE and the excised band was eluted into TE.

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. .. The ligation mixture was then used as a template for a 20-cycle PCR with the primers oKrmit-Tnseq2 and oAdapterPCR , resulting in the production of 176-bp Krmit insertion tags ( ) that were purified from a 2% agarose gel.

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. These products were ligated to double-stranded barcoded DNA adaptors using T4 DNA ligase (New England Biolabs) and purified (Qiaquick PCR Purification Kit).

    Article Title: Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn‐Seq analysis
    Article Snippet: Four micrograms of DNA from each sample were used in a Mme I (New England Biolabs, Ipswich, MA, USA) digestion reaction according to the manufacturer's instructions at 37 °C overnight. .. These reactions were purified with a QIAquick PCR purification kit (Qiagen, Germantown, MD, USA) and eluted in 60 µL of 10 m m Tris‐HCl.

    Article Title: Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa
    Article Snippet: .. Reverse transcription using oligod (T) and PCR enrichment were performed, and the PCR products were purified and digested with Mme I (New England Biolabs (NEB), Ipswich, MA, USA). ..

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. These products were ligated to double-stranded barcoded DNA adaptors using T4 DNA ligase (New England Biolabs) and purified (Qiaquick PCR Purification Kit).

    Article Title: 5? Long serial analysis of gene expression (LongSAGE) and 3? LongSAGE for transcriptome characterization and genome annotation
    Article Snippet: As in the standard SAGE procedure, to avoid PCR inhibition, the cDNA pool was first divided into two aliquots, each of which was then ligated to an adapter differing in the 5′ PCR primer annealing region. .. After size fractionation, the selected cDNA was immobilized and digested with Mme I (New England Biolabs, Beverly, MA) to release the 5′-terminal tags.

    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. .. PCR was then performed on ~80% of this purified sample using primers matching the sequences of adapter A and adapter B.

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: Double-stranded cDNA was purified by QIAquick PCR purification kit (Qiagen) and checked by electrophoresis on a 1.5% agarose gel. .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: .. Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below). .. Products were size selected using Chromaspin TE-400 columns (Clontech#636076), blunted using NEB kit (NEB# E1201L), and quantified.

    Article Title: Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1 [w]
    Article Snippet: After the beads were washed thoroughly, pools A and B were treated with 20 units of Mme I (37°C, 3 h) (New England Biolabs, Inc., Beverly, MA). .. The ligated ditag mixture was diluted (1:100 [v/v]), and 1 μL was used in a 50-μL PCR mixture.

    Quantitative RT-PCR:

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: First strand cDNA was synthesized using SuperScript™ III First-Strand Synthesis System for qRT-PCR (Invitrogen). .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation
    Article Snippet: .. It was then digested by MmeI New England Biolabs (NEB) and purified by 6% PAGE and the excised band was eluted into TE. ..

    IA:

    Article Title: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
    Article Snippet: For each sample, 1 μg of genomic DNA was sequentially digested with mapping enzyme Sac I (New England Biolabs), ligated to 20-40 ng of biotinylated linkers (Integrated DNA Technologies, Coralville, IA), and digested with the fragmenting enzyme Nla III (New England Biolabs). .. Captured DNA fragments were ligated to linkers containing Mme I recognition sites, and tags were released with Mme I (New England Biolabs).

    Article Title: Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis 1 [w]
    Article Snippet: The linker oligonucleotides, i.e. linker 1 (linker 1A [5′-TTTGGATTTGCTGGTGCAGTACAACT AGGCTTAATATCCGACATG-3′] and linker 1B [5′-TCGGATATTAAGCCTAGTTGT ACTGCACCAGCAAATCC-C7 amino-modified-3′]) and linker 2 (linker 2A [5′-TTTCT GCTCGAATTCAAGCTTCTAACGATGTACGTCCGACATG-3′ and linker 2B 5′-TC GGACGTACATCG TTAGAAGCTTGAATTCGAGCAG-C7 amino-modified-3′]) were synthesized and purified on a polyacrylamide gel (Integrated DNA Technologies Inc., Coralville, IA) as reported by Saha et al. ( ). .. After the beads were washed thoroughly, pools A and B were treated with 20 units of Mme I (37°C, 3 h) (New England Biolabs, Inc., Beverly, MA).

    Agarose Gel Electrophoresis:

    Article Title: Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes
    Article Snippet: Tn-seq was performed as originally described by van Opijnen et al . with some modifications: GAS gDNA was isolated from Krmit mutant pools and 5 µg was subjected to complete digestion with Mme I, treated with Antarctic Phosphatase (NEB), and purified by phenol/chloroform extraction. .. The ligation mixture was then used as a template for a 20-cycle PCR with the primers oKrmit-Tnseq2 and oAdapterPCR , resulting in the production of 176-bp Krmit insertion tags ( ) that were purified from a 2% agarose gel.

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Following ligation of a pair of top-Phops-oligo-17bp/T7Mme I-18bp adaptors, fragments between 250 and 650 bp were isolated from agarose gel. .. Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs).

    Article Title: Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn‐Seq analysis
    Article Snippet: Four micrograms of DNA from each sample were used in a Mme I (New England Biolabs, Ipswich, MA, USA) digestion reaction according to the manufacturer's instructions at 37 °C overnight. .. Four micrograms of DNA from each sample were used in a Mme I (New England Biolabs, Ipswich, MA, USA) digestion reaction according to the manufacturer's instructions at 37 °C overnight.

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: Double-stranded cDNA was purified by QIAquick PCR purification kit (Qiagen) and checked by electrophoresis on a 1.5% agarose gel. .. The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours.

    Mouse Assay:

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: After 48 hours, all mice looked healthy and the mouse lungs were harvested following CO2 euthanasia, weighed, homogenized, and inoculated into 25 mL LB broth. .. Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size.

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: After 48 hours, all mice looked healthy and the mouse lungs were harvested following CO2 euthanasia, weighed, homogenized, and inoculated into 25 mL LB broth. .. Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size.

    Software:

    Article Title: Genome of Paulownia (Paulownia fortunei) illuminates the related transcripts, miRNA and proteins for salt resistance
    Article Snippet: RNAs with poly (A) in the 3′adapter and a Mme I (NEB, Ipswich, MA, USA) recognition site in the 5′ adapter were isolated. .. The degradome sequencing reads with 20 and 21 nt were used in the PairFinder software to identify potentially cleaved targets .

    Real-time Polymerase Chain Reaction:

    Article Title: Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows
    Article Snippet: Primary PCR amplification of the library was conducted on a qPCR thermal cycler with a 5′ PCR primer (AAG CAG TGG TAT CAA CGC AGA GT ) and the 3′ synthesis primer. .. Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1 /Mme1 (NEB#R0637L) in a double digest (see below).

    Sample Prep:

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs). .. After treatment with Klenow Enzyme (New England Biolabs), indexed libraries were prepared with a Multiplexing Sample Preparation Oligonucleotide Kit (Illumina) according to the user's manual.

    Ethanol Precipitation:

    Article Title: Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn‐Seq analysis
    Article Snippet: Total DNA was extracted from duplicate samples of the two different growth conditions (i.e. LB and in planta ) using a Qiagen DNeasy Blood & Tissue Kit according to the manufacturer's recommendations for Gram‐negative bacteria, and then concentrated via ethanol precipitation. .. Four micrograms of DNA from each sample were used in a Mme I (New England Biolabs, Ipswich, MA, USA) digestion reaction according to the manufacturer's instructions at 37 °C overnight.

    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.

    Article Title: 5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
    Article Snippet: The cDNAs were digested by 10units of Mme I (New England Biolabs) at 37°C for 2 hours. .. Following ethanol precipitation with ammonium acetate at −70°C for 4 h, pellets were collected and resuspended in low-salt TE buffer.

    Next-Generation Sequencing:

    Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq
    Article Snippet: Then the DNA was amplified by MmeI-SD-1, MmeI-SD-2, MmeI-SD-3 and T7 primers to add a Mme I recognition site at both ends, followed by digestion with Mme I (New England Biolabs). .. The fragments between 250 and 500 bp were isolated for sequencing on an Illumina next-generation sequencing platform.

    Fractionation:

    Article Title: 5? Long serial analysis of gene expression (LongSAGE) and 3? LongSAGE for transcriptome characterization and genome annotation
    Article Snippet: .. After size fractionation, the selected cDNA was immobilized and digested with Mme I (New England Biolabs, Beverly, MA) to release the 5′-terminal tags. .. The released tags were then pooled and ligated to form 5′LS ditags that were amplified by PCR.

    DNA Purification:

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Cells were grown overnight in LB liquid, plated on plain LB agar to ensure only the expected colony morphology (i.e. no contamination occurred from gut microbiota), and 1 mL of this was subjected to genomic DNA purification as in [ ]. .. Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size.

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: Cells were grown overnight in LB liquid, plated on plain LB agar to ensure only the expected colony morphology (i.e. no contamination occurred from gut microbiota), and 1 mL of this was subjected to genomic DNA purification as in [ ]. .. Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size.

    Gel Extraction:

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: .. Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. These products were ligated to double-stranded barcoded DNA adaptors using T4 DNA ligase (New England Biolabs) and purified (Qiaquick PCR Purification Kit).

    Article Title: A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production
    Article Snippet: .. Briefly, 50 μg of genomic DNA was restricted using 50U Mme I (New England Biolabs) overnight at 37 degrees followed by gel extraction (Qiagen Gel Extraction Kit) to excise a band 1.7–2.1 kb in size. .. These products were ligated to double-stranded barcoded DNA adaptors using T4 DNA ligase (New England Biolabs) and purified (Qiaquick PCR Purification Kit).

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  • 95
    New England Biolabs mmei
    Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The <t>DNA</t> is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by <t>MmeI</t> to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.
    Mmei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mmei/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mmei - by Bioz Stars, 2020-02
    95/100 stars
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    84
    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: 84/100, 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
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    mmei site - by Bioz Stars, 2020-02
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    Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.

    Journal: Nucleic Acids Research

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

    doi: 10.1093/nar/gkl391

    Figure Lengend Snippet: Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.

    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).

    Techniques: Amplification, Blocking Assay, Generated, Magnetic Beads, Polymerase Chain Reaction

    Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

    Journal: Nature Communications

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

    doi: 10.1038/s41467-019-13719-9

    Figure Lengend Snippet: Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

    Article Snippet: Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol.

    Techniques: Isolation, Mutagenesis, Amplification, Produced, Sequencing

    Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

    Journal: Nature Communications

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

    doi: 10.1038/s41467-019-13719-9

    Figure Lengend Snippet: Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

    Article Snippet: Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol.

    Techniques: Whole Genome Amplification, Sequencing, Amplification, Ligation

    Proof-of-principle experiment of RLL-Y2H system. ( A ) Positive control interacting protein pair, murine p53 and SV40 T-antigen were inserted into mBD and mAD of RLL-Y2H system and were transformed into yeast respectively. Only the yeasts containing both p53 and SV40 T-antigen but not the controls can grow on SD/-AHL T selection plates. ( B ) After plasmid extraction from yeast, p53, SV40 T-antigen and their recombinated fragments were amplified by PCR. ( C ) Sanger sequencing of the recombinated p53 and SV40 T-antigen fragments. The linker ATTL and MmeI sites were highlighted. ( D ) The PCR products of the recombinated p53 and SV40 T-antigen fragments before (left panel) and after (right panel) MmeI digestion.

    Journal: Nucleic Acids Research

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system

    doi: 10.1093/nar/gkx1173

    Figure Lengend Snippet: Proof-of-principle experiment of RLL-Y2H system. ( A ) Positive control interacting protein pair, murine p53 and SV40 T-antigen were inserted into mBD and mAD of RLL-Y2H system and were transformed into yeast respectively. Only the yeasts containing both p53 and SV40 T-antigen but not the controls can grow on SD/-AHL T selection plates. ( B ) After plasmid extraction from yeast, p53, SV40 T-antigen and their recombinated fragments were amplified by PCR. ( C ) Sanger sequencing of the recombinated p53 and SV40 T-antigen fragments. The linker ATTL and MmeI sites were highlighted. ( D ) The PCR products of the recombinated p53 and SV40 T-antigen fragments before (left panel) and after (right panel) MmeI digestion.

    Article Snippet: Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification.

    Techniques: Positive Control, Transformation Assay, Selection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Sequencing

    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