buffer 4  (New England Biolabs)


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  • 99
    Name:
    AluI
    Description:
    AluI 5 000 units
    Catalog Number:
    R0137L
    Price:
    277
    Size:
    5 000 units
    Category:
    Restriction Enzymes
    Score:
    85
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    Structured Review

    New England Biolabs buffer 4
    AluI
    AluI 5 000 units
    https://www.bioz.com/result/buffer 4/product/New England Biolabs
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    buffer 4 - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands"

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp046

    Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42,  32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t  or S b ) and cleaved strands (P 16  or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1  and 0.3 min −1  for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1  and 0.5 min −1  for top and bottom strands, respectively.
    Figure Legend Snippet: Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42, 32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t or S b ) and cleaved strands (P 16 or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1 and 0.3 min −1 for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1 and 0.5 min −1 for top and bottom strands, respectively.

    Techniques Used: Selection, Polyacrylamide Gel Electrophoresis

    Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM  3 H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. ( A ) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. ( B ) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.
    Figure Legend Snippet: Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM 3 H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. ( A ) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. ( B ) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.

    Techniques Used: Concentration Assay

    Strand-specific nicking. ( A ) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was  32 P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S t  and S b , from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P 16  from the top strand, P 12  from the bottom). ( B ) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min −1 . Data points for the bottom strand are connected by a dashed line.
    Figure Legend Snippet: Strand-specific nicking. ( A ) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was 32 P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S t and S b , from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P 16 from the top strand, P 12 from the bottom). ( B ) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min −1 . Data points for the bottom strand are connected by a dashed line.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. ( A ) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. ( B ) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.
    Figure Legend Snippet: Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. ( A ) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. ( B ) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.

    Techniques Used: Plasmid Preparation, Electrophoresis, Concentration Assay

    2) Product Images from "A quantitative understanding of lac repressor’s binding specificity and flexibility"

    Article Title: A quantitative understanding of lac repressor’s binding specificity and flexibility

    Journal:

    doi: 10.1007/s40484-015-0044-z

    Specificity profiling the the whole lac operator. (A) Energy logo for the whole lac operator under 1× NEB buffer 4 condition. Energy logo was produced based on the linear regression of all the wild-type O1 operator’s single and adjacent double variants’ energy values. (B) Measured vs. predicted energy deviation for all double variant pairs. The energy value of each adjacent double variant can either be measured directly or predicted by summing up the contributions of each individual base variation. Each double variant is plotted in panel B based on its position in the operator and energy deviation value between measured and predicted numbers. (C) Distribution of measured vs. predicted energy for all double variants. All energy deviation values in panel B were re-plotted in histogram form, and it is clear that most of adjacent double variants follow “additivity assumption” reasonably well.
    Figure Legend Snippet: Specificity profiling the the whole lac operator. (A) Energy logo for the whole lac operator under 1× NEB buffer 4 condition. Energy logo was produced based on the linear regression of all the wild-type O1 operator’s single and adjacent double variants’ energy values. (B) Measured vs. predicted energy deviation for all double variant pairs. The energy value of each adjacent double variant can either be measured directly or predicted by summing up the contributions of each individual base variation. Each double variant is plotted in panel B based on its position in the operator and energy deviation value between measured and predicted numbers. (C) Distribution of measured vs. predicted energy for all double variants. All energy deviation values in panel B were re-plotted in histogram form, and it is clear that most of adjacent double variants follow “additivity assumption” reasonably well.

    Techniques Used: Produced, Variant Assay

    Related Articles

    Diagnostic Assay:

    Article Title: Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests
    Article Snippet: PCR amplification of this diagnostic barcode region (476–482 bp) was performed with the primers Cat0 and Nancy [ ] labeled with HEX and ATTO565 dyes, respectively ( ). .. PCR products were subsequently cut overnight at 37 °C in 20 µL reaction volumes with one unit of the AluI (NEB, Ipswich, MA, USA) restriction enzyme.

    Clone Assay:

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany).

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia
    Article Snippet: These double digests were performed in a 20 μl reaction containing 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 unit Fsp I and 1 unit Alu I (New England Biolabs, Ipswich, MA, USA), and incubated at 37°C for 4 hours or overnight. .. These double digests were performed in a 20 μl reaction containing 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 unit Fsp I and 1 unit Alu I (New England Biolabs, Ipswich, MA, USA), and incubated at 37°C for 4 hours or overnight.

    Centrifugation:

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ).

    Amplification:

    Article Title: Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout
    Article Snippet: Twenty microliters of RCA mixture containing 0.2 µg/µl BSA, 125 µM dNTP (Fermentas), 1× Phi29 buffer (Fermentas) and 100 mU/µl Phi29 DNA polymerase (Fermentas) was added to the Dynabeads. .. RCA was performed at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. To monomerize the RCA products, restriction oligonucleotide L12890 (120 nM) was added with 0.2 µg/µl BSA and 120 mU/µl of Alu I (New England Biolabs) in 1× Phi29 buffer followed by incubation at 37°C for 1 min and enzyme inactivation at 65°C for 1 min. After the beads were discarded, the monomerized RCA products were re-circularized and amplified by addition of 1× Phi29 buffer, 0.2 µg/µl BSA, 100 µM dNTP, 0.68 mM ATP, 60 mU/µl Phi29 DNA polymerase, 14 mU/µl T4 DNA ligase (Fermentas) and incubated at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. .. Initial probe design was compared to improved oligonucleotides by applying different sets of wild type probe components on 10 amol synthetic target DNA (L10919).

    Article Title: A functional polymorphism in the IL1B gene promoter, IL1B -31C > T, is not associated with cerebral malaria in Thailand
    Article Snippet: For the IL1B promoter polymorphism, IL1B -31C > T [ ], PCR was carried out with primers IL1B5'F (5'-TAGTCCCCTCCCCTAAGAACG-3') and IL1Bint1R (5'-CCCAGAATATTTCCCGAGTCA-3') to amplify the region including IL1B -31C > T, and the PCR products were treated with Alu I (New England Biolabs, Beverly, CA, USA), which digests the -31T allele. .. For the IL1B exon 5 polymorphism, IL1B 3953C>T [ ], PCR was carried out with primers IL1Bint4F (5'-GCTCAGGTGTCCTCCAAGAAA-3') and IL1Bint5R (5'-GGCCAGTGCAATCAAATGTG-3'), and the PCR products were treated with Taq I (New England Biolabs), which digests the 3953C allele.

    Article Title: iGentifier: indexing and large-scale profiling of unknown transcriptomes
    Article Snippet: Digesting RNA preparations with DNase I proved to be not required and thus was omitted from our protocol. .. cDNA synthesis, fragmentation and amplification were performed according to ( ) with the following exceptions: fragmentation was achieved with 20 U of AluI (New England Biolabs); and ligation of blunt-ended linker DAL4138 (upper strand: 5′-TGGATAGAGCAGTGGTAGCACACGTGAGCGATGACTATGAG-3′, lower strand: 5′-CTCATAGTCATCGCTCACGTGTCGGCACATCTCATATA-3′) was performed with 1 U of T4 DNA Quick ligase (New England Biolabs) in the reaction buffer. .. PCR was executed with 1 U of Taq polymerase (Invitrogen) pre-incubated with TaqStart antibody (Clontech) in a total volume of 20 μl using microtitre plates and PE 9700 thermocyclers (Perkin-Elmer).

    Article Title: The paratransgenic sand fly: A platform for control of Leishmania transmission
    Article Snippet: Bacteria were initially identified using amplified ribosomal DNA restriction analysis (ARDRA) [ ]. .. The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels.

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). .. After incubating for 2 h at 37 °C, 5 mM EDTA was added to terminate the reaction.

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol. .. DNA probe was made by isolating the target satellite sequence using the PCR reaction and the primers Forward (5′-CGG-TTA-TTT-CTG-TGT-TGT-TTC-GG-3′) and Reverse (5′-TGA-CCA-GTC-TGC-AGC-AAA-AC-3′) which were specifically designed for this purpose.

    Article Title: Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests
    Article Snippet: PCR amplification of this diagnostic barcode region (476–482 bp) was performed with the primers Cat0 and Nancy [ ] labeled with HEX and ATTO565 dyes, respectively ( ). .. PCR products were subsequently cut overnight at 37 °C in 20 µL reaction volumes with one unit of the AluI (NEB, Ipswich, MA, USA) restriction enzyme.

    Article Title: The association between MMP-1 gene rs1799750 polymorphism and knee osteoarthritis risk
    Article Snippet: Amplification was performed using a PTC-100 thermal cycler (MJ Research, Inc.). .. The PCR cycling conditions were 2 min at 95°C followed by 35 cycles of 45 s at 94°C, 60 s at 49.5°C (MMP1), and 60 s at 72°C, and with a final extension at 72°C for 10 min. PCR products were digested for 16 h at 37°C (MMP-1) in a 15 ml reaction containing 5 U AluI restriction enzyme (New England Biolabs) for determination of MMP-1 genotypes.

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia
    Article Snippet: As such, amplified ITS2 products (50 to 100 ng/μl) were selected for the enzyme restriction analysis and digested according to manufacturer’s instructions (Promega, Madison, WI and New England Biolabs, Ipswich, MA, USA). .. These double digests were performed in a 20 μl reaction containing 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 unit Fsp I and 1 unit Alu I (New England Biolabs, Ipswich, MA, USA), and incubated at 37°C for 4 hours or overnight.

    Article Title: Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie
    Article Snippet: Universal primers (forward, 5′-AGAGTTTGATCATGGCTCAG-3′; and reverse, 5′-GGTTACCTTGTTACGACTT-3′) were used to PCR amplify a 1,465-bp 16S rRNA gene segment, using GoTaq Green master mix (Promega, Madison, WI). .. The amplicon was subsequently double digested with AluI and MboI (New England BioLabs, Ipswich, MA) at 37°C overnight and electrophoresed in a 4% MetaPhor agarose gel (Lonza, Rockland, ME) as previously described ( ). .. Strains of A. hydrophila (ATCC 7966), A. caviae (ATCC 15468), and A. veronii (ATCC 9071) were used as positive controls.

    Whole Genome Amplification:

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). .. After incubating for 2 h at 37 °C, 5 mM EDTA was added to terminate the reaction.

    Terminal Restriction Fragment Length Polymorphism:

    Article Title: Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil
    Article Snippet: Paragraph title: TRFLP and data analysis. ... Thirty nanograms of carboxyfluorescein-labeled PCR products were independently digested using 2 units of the 4-base-pair-cutting restriction enzyme MnlI or AluI (NEB, Beverly, MA).

    Electrophoresis:

    Article Title: A functional polymorphism in the IL1B gene promoter, IL1B -31C > T, is not associated with cerebral malaria in Thailand
    Article Snippet: For the IL1B promoter polymorphism, IL1B -31C > T [ ], PCR was carried out with primers IL1B5'F (5'-TAGTCCCCTCCCCTAAGAACG-3') and IL1Bint1R (5'-CCCAGAATATTTCCCGAGTCA-3') to amplify the region including IL1B -31C > T, and the PCR products were treated with Alu I (New England Biolabs, Beverly, CA, USA), which digests the -31T allele. .. For the IL-1RA VNTR polymorphism [ ], the region including variable numbers of identical 86-bp tandem repeats was amplified by PCR using the following primers: 5'-CTCAGCCAACACTCCTAT-3' and 5'-TCCTGGTCTGCAGGTAA-3'.

    Microarray:

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Microarray analysis. .. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ).

    Incubation:

    Article Title: Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout
    Article Snippet: Twenty microliters of RCA mixture containing 0.2 µg/µl BSA, 125 µM dNTP (Fermentas), 1× Phi29 buffer (Fermentas) and 100 mU/µl Phi29 DNA polymerase (Fermentas) was added to the Dynabeads. .. RCA was performed at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. To monomerize the RCA products, restriction oligonucleotide L12890 (120 nM) was added with 0.2 µg/µl BSA and 120 mU/µl of Alu I (New England Biolabs) in 1× Phi29 buffer followed by incubation at 37°C for 1 min and enzyme inactivation at 65°C for 1 min. After the beads were discarded, the monomerized RCA products were re-circularized and amplified by addition of 1× Phi29 buffer, 0.2 µg/µl BSA, 100 µM dNTP, 0.68 mM ATP, 60 mU/µl Phi29 DNA polymerase, 14 mU/µl T4 DNA ligase (Fermentas) and incubated at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. .. Initial probe design was compared to improved oligonucleotides by applying different sets of wild type probe components on 10 amol synthetic target DNA (L10919).

    Article Title: Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia
    Article Snippet: For positive samples, RFLP on PCR products using AluI enzyme was performed for determining Plasmodium species. .. Briefly, 5 µl of PCR products adjusted to 500 ng/µl were mixed with 0.2 µl of AluI and 2.5 µl of buffer A according to the manufacturer's instructions (New England Biolabs®, France), incubated for 4 hours at 37°C and inactivated at 65°C for 15 minutes. .. Bands were detected by standard 2% agarose gel electrophoresis and ethidium bromide staining.

    Article Title: MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans
    Article Snippet: AluI cleaves the PCR product from a wild-type sequence once but does not cut the mutant sequence. .. PCR product (10 μl) was incubated with 10 U AluI (New England BioLabs) in a 30-μl reaction at 37°C for 2 hours. .. Digestion products were resolved on a 2% agarose gel.

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia
    Article Snippet: In addition to single restriction digests, double restriction digests with Alu I/ Fsp I were used to discriminate An. nuneztovari s.l. from An. benarrochi s.l. ( ; ). .. These double digests were performed in a 20 μl reaction containing 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 unit Fsp I and 1 unit Alu I (New England Biolabs, Ipswich, MA, USA), and incubated at 37°C for 4 hours or overnight. .. Positive controls corresponding to the ITS2 digest of each species being analyzed were included in every gel.

    Article Title: Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation
    Article Snippet: Hoechst staining was performed as described ( ). .. Isolated nuclei from 1 × 107 cells were incubated overnight at 37 °C in 100 μl of AluI buffer (New England Biolabs; 50 m m NaCl, 10 m m Tris-Cl, 10 m m MgCl2 , 1 m m DTT, pH 7.9) with 1 unit/μl of AluI. .. The nuclei incubated in the same buffer without AluI served as a negative control.

    Article Title: ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner
    Article Snippet: Cells were resuspended in proteinase K lysis buffer (10 mM Tris-HCl, pH 8; 100 mM EDTA; 0.5% SDS; 20 μg/mL RNase A; 100 μg/mL proteinase K) and incubated at 37°C with agitation for five hours. .. Two equal volume phenol:chloroform:isoamyl alcohol (25:24:1, pH 8) extractions were performed, followed by two ethanol precipitations and final resuspension in H2 O. DNA was digested overnight with AluI and MboI (New England Biolabs).

    Expressing:

    Article Title: The paratransgenic sand fly: A platform for control of Leishmania transmission
    Article Snippet: The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels. .. The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels.

    Transformation Assay:

    Article Title: The paratransgenic sand fly: A platform for control of Leishmania transmission
    Article Snippet: The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels. .. The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels.

    Hybridization:

    Article Title: Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout
    Article Snippet: Hybridization and ligation were performed at 60°C for 5 min. MyOne Dynabeads T1 (Invitrogen) were washed three times in 1× Wtw buffer (10 mM TRIS-HCl pH 7.5, 5 mM EDTA, 0.1% Tween 20 [Sigma-Aldrich], 0.1 M NaCl). .. RCA was performed at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. To monomerize the RCA products, restriction oligonucleotide L12890 (120 nM) was added with 0.2 µg/µl BSA and 120 mU/µl of Alu I (New England Biolabs) in 1× Phi29 buffer followed by incubation at 37°C for 1 min and enzyme inactivation at 65°C for 1 min. After the beads were discarded, the monomerized RCA products were re-circularized and amplified by addition of 1× Phi29 buffer, 0.2 µg/µl BSA, 100 µM dNTP, 0.68 mM ATP, 60 mU/µl Phi29 DNA polymerase, 14 mU/µl T4 DNA ligase (Fermentas) and incubated at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min.

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: Paragraph title: Southern blot hybridization ... For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol.

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. DNA concentration was determined spectrophotometrically and samples were stored at −80°C.

    High Performance Liquid Chromatography:

    Article Title: Chemical mapping of cytosines enzymatically flipped out of the DNA helix
    Article Snippet: M.AluI (50 units/μl) and M.SssI (4 units/μl) were purchased from New England Biolabs (USA). .. Restriction endonucleases R.Ecl18kI, R.PspGI and R.BcnI were purified and their concentrations were determined as described ( , ).

    Gas Chromatography:

    Article Title: Chemical mapping of cytosines enzymatically flipped out of the DNA helix
    Article Snippet: M.AluI (50 units/μl) and M.SssI (4 units/μl) were purchased from New England Biolabs (USA). .. Restriction endonucleases R.Ecl18kI, R.PspGI and R.BcnI were purified and their concentrations were determined as described ( , ).

    Southern Blot:

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: Paragraph title: Southern blot hybridization ... For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol.

    Article Title: ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner
    Article Snippet: Paragraph title: TRF Southern blotting ... Two equal volume phenol:chloroform:isoamyl alcohol (25:24:1, pH 8) extractions were performed, followed by two ethanol precipitations and final resuspension in H2 O. DNA was digested overnight with AluI and MboI (New England Biolabs).

    Ligation:

    Article Title: Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout
    Article Snippet: Paragraph title: Padlock Probe Ligation and Amplification ... RCA was performed at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. To monomerize the RCA products, restriction oligonucleotide L12890 (120 nM) was added with 0.2 µg/µl BSA and 120 mU/µl of Alu I (New England Biolabs) in 1× Phi29 buffer followed by incubation at 37°C for 1 min and enzyme inactivation at 65°C for 1 min. After the beads were discarded, the monomerized RCA products were re-circularized and amplified by addition of 1× Phi29 buffer, 0.2 µg/µl BSA, 100 µM dNTP, 0.68 mM ATP, 60 mU/µl Phi29 DNA polymerase, 14 mU/µl T4 DNA ligase (Fermentas) and incubated at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min.

    Article Title: iGentifier: indexing and large-scale profiling of unknown transcriptomes
    Article Snippet: Digesting RNA preparations with DNase I proved to be not required and thus was omitted from our protocol. .. cDNA synthesis, fragmentation and amplification were performed according to ( ) with the following exceptions: fragmentation was achieved with 20 U of AluI (New England Biolabs); and ligation of blunt-ended linker DAL4138 (upper strand: 5′-TGGATAGAGCAGTGGTAGCACACGTGAGCGATGACTATGAG-3′, lower strand: 5′-CTCATAGTCATCGCTCACGTGTCGGCACATCTCATATA-3′) was performed with 1 U of T4 DNA Quick ligase (New England Biolabs) in the reaction buffer. .. PCR was executed with 1 U of Taq polymerase (Invitrogen) pre-incubated with TaqStart antibody (Clontech) in a total volume of 20 μl using microtitre plates and PE 9700 thermocyclers (Perkin-Elmer).

    Article Title: Single-Cell Semiconductor Sequencing
    Article Snippet: Alu I and NEB buffer 4 (Cat # R0137S, New England BioLabs). .. Alu I and NEB buffer 4 (Cat # R0137S, New England BioLabs).

    Cell Culture:

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany).

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: JC326 cells were cultured overnight in M9+glucose (0.2%) medium ( ) that also contained tetracycline (25 μg/ml). .. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ).

    Generated:

    Article Title: iGentifier: indexing and large-scale profiling of unknown transcriptomes
    Article Snippet: cDNA synthesis, fragmentation and amplification were performed according to ( ) with the following exceptions: fragmentation was achieved with 20 U of AluI (New England Biolabs); and ligation of blunt-ended linker DAL4138 (upper strand: 5′-TGGATAGAGCAGTGGTAGCACACGTGAGCGATGACTATGAG-3′, lower strand: 5′-CTCATAGTCATCGCTCACGTGTCGGCACATCTCATATA-3′) was performed with 1 U of T4 DNA Quick ligase (New England Biolabs) in the reaction buffer. .. cDNA synthesis, fragmentation and amplification were performed according to ( ) with the following exceptions: fragmentation was achieved with 20 U of AluI (New England Biolabs); and ligation of blunt-ended linker DAL4138 (upper strand: 5′-TGGATAGAGCAGTGGTAGCACACGTGAGCGATGACTATGAG-3′, lower strand: 5′-CTCATAGTCATCGCTCACGTGTCGGCACATCTCATATA-3′) was performed with 1 U of T4 DNA Quick ligase (New England Biolabs) in the reaction buffer.

    Article Title: Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil
    Article Snippet: Thirty nanograms of carboxyfluorescein-labeled PCR products were independently digested using 2 units of the 4-base-pair-cutting restriction enzyme MnlI or AluI (NEB, Beverly, MA). .. Samples were then denatured at 95°C for 5 min, and the lengths of the individual restriction fragments were determined using an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA) to determine their TRFLP patterns.

    DNA Labeling:

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany).

    Polymerase Chain Reaction:

    Article Title: A functional polymorphism in the IL1B gene promoter, IL1B -31C > T, is not associated with cerebral malaria in Thailand
    Article Snippet: Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp Blood Kit (Qiagen, Hilden, Germany). .. For the IL1B promoter polymorphism, IL1B -31C > T [ ], PCR was carried out with primers IL1B5'F (5'-TAGTCCCCTCCCCTAAGAACG-3') and IL1Bint1R (5'-CCCAGAATATTTCCCGAGTCA-3') to amplify the region including IL1B -31C > T, and the PCR products were treated with Alu I (New England Biolabs, Beverly, CA, USA), which digests the -31T allele. .. For the IL1B exon 5 polymorphism, IL1B 3953C>T [ ], PCR was carried out with primers IL1Bint4F (5'-GCTCAGGTGTCCTCCAAGAAA-3') and IL1Bint5R (5'-GGCCAGTGCAATCAAATGTG-3'), and the PCR products were treated with Taq I (New England Biolabs), which digests the 3953C allele.

    Article Title: The paratransgenic sand fly: A platform for control of Leishmania transmission
    Article Snippet: Cells were lysed using an initial cycle step of 94°C for 2 mins. .. The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels. .. The banding pattern of each sample is compared to that of a known B. subtilis control.

    Article Title: Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia
    Article Snippet: For positive samples, RFLP on PCR products using AluI enzyme was performed for determining Plasmodium species. .. Briefly, 5 µl of PCR products adjusted to 500 ng/µl were mixed with 0.2 µl of AluI and 2.5 µl of buffer A according to the manufacturer's instructions (New England Biolabs®, France), incubated for 4 hours at 37°C and inactivated at 65°C for 15 minutes. .. Bands were detected by standard 2% agarose gel electrophoresis and ethidium bromide staining.

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). .. DNA was ethanol precipitated, resuspended in 10 μL ddH2O, and then conjugated to fluorophores using ARES Alexa Fluor DNA labeling kits (A-21665, A21667, and A-21676; Life Technologies) for 2 h, according to the manufacturer’s instructions.

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol. .. DNA probe was made by isolating the target satellite sequence using the PCR reaction and the primers Forward (5′-CGG-TTA-TTT-CTG-TGT-TGT-TTC-GG-3′) and Reverse (5′-TGA-CCA-GTC-TGC-AGC-AAA-AC-3′) which were specifically designed for this purpose.

    Article Title: Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests
    Article Snippet: PCR amplifications were performed in 12 µL reaction volumes containing 10 mM Tris-HCl, pH 9, 50 mM KCl, 200 µM of each dNTP, 0.4 µM of each primer, 1.5 mM MgCl2 , one unit Taq DNA polymerase (Promega), 0.1 mg/mL BSA with 2 µL of DNA template. .. PCR products were subsequently cut overnight at 37 °C in 20 µL reaction volumes with one unit of the AluI (NEB, Ipswich, MA, USA) restriction enzyme. .. Digested fragments were visualized after electrophoresis on an ABI3730 DNA sequencer.

    Article Title: The association between MMP-1 gene rs1799750 polymorphism and knee osteoarthritis risk
    Article Snippet: Amplification was performed using a PTC-100 thermal cycler (MJ Research, Inc.). .. The PCR cycling conditions were 2 min at 95°C followed by 35 cycles of 45 s at 94°C, 60 s at 49.5°C (MMP1), and 60 s at 72°C, and with a final extension at 72°C for 10 min. PCR products were digested for 16 h at 37°C (MMP-1) in a 15 ml reaction containing 5 U AluI restriction enzyme (New England Biolabs) for determination of MMP-1 genotypes. .. The digested products were subjected to gel electrophoresis and visualized by ethidium bromide staining.

    Article Title: PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation
    Article Snippet: Fragments were separated on a 4% NuSieve agarose gel (BioWhittaker Molecular Applications, Rockland, Maine) at constant current in Tris-borate-EDTA buffer for approximately 1.5 h. The agarose gels, containing ethidium bromide (0.5 μg/ml), were photographed under UV light with a gel documentation system (UVP ImageStore 5000; Ultra-Violet Products Ltd., Cambridge, United Kingdom). .. The positive PCR products from the second reaction (10 μl) were digested with 1 U of Alu I (New England Biolabs) at 37°C for 2 h. Fragments were separated, visualized, and photographed as described above. .. During our initial SAR monitoring study, we detected five samples that were PCR-RFLP positive for C . cayetanensis as described by Relman et al. ( ) and Jinneman et al. ( ).

    Article Title: MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans
    Article Snippet: AluI cleaves the PCR product from a wild-type sequence once but does not cut the mutant sequence. .. PCR product (10 μl) was incubated with 10 U AluI (New England BioLabs) in a 30-μl reaction at 37°C for 2 hours. .. Digestion products were resolved on a 2% agarose gel.

    Article Title: Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil
    Article Snippet: A 15-min extension step at 72°C was applied after PCR amplification. .. Thirty nanograms of carboxyfluorescein-labeled PCR products were independently digested using 2 units of the 4-base-pair-cutting restriction enzyme MnlI or AluI (NEB, Beverly, MA). .. Samples were then denatured at 95°C for 5 min, and the lengths of the individual restriction fragments were determined using an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA) to determine their TRFLP patterns.

    Article Title: Single-Cell Semiconductor Sequencing
    Article Snippet: SMARTer® PCR cDNA Synthesis Kit (Clontech Cat # 634926). .. Alu I and NEB buffer 4 (Cat # R0137S, New England BioLabs).

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia
    Article Snippet: Paragraph title: 2.3. PCR-RFLP-ITS2 analysis ... These double digests were performed in a 20 μl reaction containing 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 unit Fsp I and 1 unit Alu I (New England Biolabs, Ipswich, MA, USA), and incubated at 37°C for 4 hours or overnight.

    Article Title: Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie
    Article Snippet: Universal primers (forward, 5′-AGAGTTTGATCATGGCTCAG-3′; and reverse, 5′-GGTTACCTTGTTACGACTT-3′) were used to PCR amplify a 1,465-bp 16S rRNA gene segment, using GoTaq Green master mix (Promega, Madison, WI). .. The amplicon was subsequently double digested with AluI and MboI (New England BioLabs, Ipswich, MA) at 37°C overnight and electrophoresed in a 4% MetaPhor agarose gel (Lonza, Rockland, ME) as previously described ( ).

    Article Title: Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation
    Article Snippet: Isolated nuclei from 1 × 107 cells were incubated overnight at 37 °C in 100 μl of AluI buffer (New England Biolabs; 50 m m NaCl, 10 m m Tris-Cl, 10 m m MgCl2 , 1 m m DTT, pH 7.9) with 1 unit/μl of AluI. .. DNA was isolated by extraction with a phenol-chloroform-isoamyl alcohol solution and ethanol precipitation and dissolved in 500 μl of 10 m m TrisCl, 1 m m Na2 EDTA, pH 7.9.

    DNA Extraction:

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: DNA was extracted from E. tef var Enatite seedlings, grown as described in “Plant material and DNA extraction”. .. For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol.

    Article Title: The association between MMP-1 gene rs1799750 polymorphism and knee osteoarthritis risk
    Article Snippet: Paragraph title: DNA extraction and genotyping ... The PCR cycling conditions were 2 min at 95°C followed by 35 cycles of 45 s at 94°C, 60 s at 49.5°C (MMP1), and 60 s at 72°C, and with a final extension at 72°C for 10 min. PCR products were digested for 16 h at 37°C (MMP-1) in a 15 ml reaction containing 5 U AluI restriction enzyme (New England Biolabs) for determination of MMP-1 genotypes.

    Fluorescence:

    Article Title: Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil
    Article Snippet: Thirty nanograms of carboxyfluorescein-labeled PCR products were independently digested using 2 units of the 4-base-pair-cutting restriction enzyme MnlI or AluI (NEB, Beverly, MA). .. Samples were then denatured at 95°C for 5 min, and the lengths of the individual restriction fragments were determined using an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA) to determine their TRFLP patterns.

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. Fluorescent labelling of DNA samples and microarray hybridization were performed, as described previously ( ).

    Mutagenesis:

    Article Title: MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans
    Article Snippet: AluI cleaves the PCR product from a wild-type sequence once but does not cut the mutant sequence. .. PCR product (10 μl) was incubated with 10 U AluI (New England BioLabs) in a 30-μl reaction at 37°C for 2 hours.

    Article Title: MYB36 regulates the transition from proliferation to differentiation in the Arabidopsis root
    Article Snippet: SNPtrack was used to analyze the data and determine the causative mutation ( ). dCAPS genotyping primers were designed ( ) ( ) to screen for a mutant allele: genotype forward (F)/reverse (R): 5′-AGATGTGGTAAGAGTTGCAGACTGAGATA-3′, 5′-caagagagatagatcaccgacCG-3′. .. Product size is 144 bp; wild type is cut into 114- and 31-bp fragments with AluI enzyme (New England Biolabs; R0137S).

    Isolation:

    Article Title: The association between MMP-1 gene rs1799750 polymorphism and knee osteoarthritis risk
    Article Snippet: Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). .. The PCR cycling conditions were 2 min at 95°C followed by 35 cycles of 45 s at 94°C, 60 s at 49.5°C (MMP1), and 60 s at 72°C, and with a final extension at 72°C for 10 min. PCR products were digested for 16 h at 37°C (MMP-1) in a 15 ml reaction containing 5 U AluI restriction enzyme (New England Biolabs) for determination of MMP-1 genotypes.

    Article Title: Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation
    Article Snippet: Hoechst staining was performed as described ( ). .. Isolated nuclei from 1 × 107 cells were incubated overnight at 37 °C in 100 μl of AluI buffer (New England Biolabs; 50 m m NaCl, 10 m m Tris-Cl, 10 m m MgCl2 , 1 m m DTT, pH 7.9) with 1 unit/μl of AluI. .. The nuclei incubated in the same buffer without AluI served as a negative control.

    Microscopy:

    Article Title: The paratransgenic sand fly: A platform for control of Leishmania transmission
    Article Snippet: The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels. .. The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels.

    Purification:

    Article Title: Chemical mapping of cytosines enzymatically flipped out of the DNA helix
    Article Snippet: M.AluI (50 units/μl) and M.SssI (4 units/μl) were purchased from New England Biolabs (USA). .. M.AluI (50 units/μl) and M.SssI (4 units/μl) were purchased from New England Biolabs (USA).

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). .. After incubating for 2 h at 37 °C, 5 mM EDTA was added to terminate the reaction.

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol. .. DNA probe was made by isolating the target satellite sequence using the PCR reaction and the primers Forward (5′-CGG-TTA-TTT-CTG-TGT-TGT-TTC-GG-3′) and Reverse (5′-TGA-CCA-GTC-TGC-AGC-AAA-AC-3′) which were specifically designed for this purpose.

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Sodium azide was added to a final concentration of 0.2% (w/v), and cells were collected by centrifugation (5,000 g , 10 min at 4°C). .. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. DNA concentration was determined spectrophotometrically and samples were stored at −80°C.

    Sequencing:

    Article Title: Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests
    Article Snippet: The Cat0 sequence was established to match the barcode sequences of both the tortricid moths and their parasitoids, and to avoid the presence of an AluI restriction site within the primer. .. PCR products were subsequently cut overnight at 37 °C in 20 µL reaction volumes with one unit of the AluI (NEB, Ipswich, MA, USA) restriction enzyme.

    Article Title: MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans
    Article Snippet: AluI cleaves the PCR product from a wild-type sequence once but does not cut the mutant sequence. .. PCR product (10 μl) was incubated with 10 U AluI (New England BioLabs) in a 30-μl reaction at 37°C for 2 hours.

    Article Title: Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie
    Article Snippet: Paragraph title: 16S rRNA gene RFLP and gyrB sequence analysis. ... The amplicon was subsequently double digested with AluI and MboI (New England BioLabs, Ipswich, MA) at 37°C overnight and electrophoresed in a 4% MetaPhor agarose gel (Lonza, Rockland, ME) as previously described ( ).

    Labeling:

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol. .. The expected amplified band was extracted and purified using Wizard SV Gel and PCR Clean-up System (Promega).

    Article Title: Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests
    Article Snippet: PCR amplification of this diagnostic barcode region (476–482 bp) was performed with the primers Cat0 and Nancy [ ] labeled with HEX and ATTO565 dyes, respectively ( ). .. PCR products were subsequently cut overnight at 37 °C in 20 µL reaction volumes with one unit of the AluI (NEB, Ipswich, MA, USA) restriction enzyme.

    Nested PCR:

    Article Title: Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia
    Article Snippet: Paragraph title: Nested PCR detection ... Briefly, 5 µl of PCR products adjusted to 500 ng/µl were mixed with 0.2 µl of AluI and 2.5 µl of buffer A according to the manufacturer's instructions (New England Biolabs®, France), incubated for 4 hours at 37°C and inactivated at 65°C for 15 minutes.

    Plasmid Preparation:

    Article Title: The paratransgenic sand fly: A platform for control of Leishmania transmission
    Article Snippet: The PCR products were digested separately with Alu I (NEB) and Taq I (NEB), and were separated on 2% agarose gels. .. GFP expression in transformed B. subtilis was verified by visualization on a Zeiss AxioSkop fluorescent microscope.

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany).

    Software:

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. Fluorescent labelling of DNA samples and microarray hybridization were performed, as described previously ( ).

    Agarose Gel Electrophoresis:

    Article Title: A functional polymorphism in the IL1B gene promoter, IL1B -31C > T, is not associated with cerebral malaria in Thailand
    Article Snippet: For the IL1B promoter polymorphism, IL1B -31C > T [ ], PCR was carried out with primers IL1B5'F (5'-TAGTCCCCTCCCCTAAGAACG-3') and IL1Bint1R (5'-CCCAGAATATTTCCCGAGTCA-3') to amplify the region including IL1B -31C > T, and the PCR products were treated with Alu I (New England Biolabs, Beverly, CA, USA), which digests the -31T allele. .. For the IL-1RA VNTR polymorphism [ ], the region including variable numbers of identical 86-bp tandem repeats was amplified by PCR using the following primers: 5'-CTCAGCCAACACTCCTAT-3' and 5'-TCCTGGTCTGCAGGTAA-3'.

    Article Title: Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome
    Article Snippet: For each enzymatic reaction, 5 μg of DNA was individually digested with the following restriction endonucleases: Xba I (R0145S; New England BioLabs), Eco RI (R0101S; New England BioLabs), Hpa II (R0171S; New England BioLabs), Msp I (R0106S; New England BioLabs) and Alu I (R0137S; New England BioLabs) following the manufacturer’s protocol. .. It was then diluted in 1:200 and used for labeling reactions by polymerase chain reaction (PCR) using DIG-11-dUTP labeling (Roche).

    Article Title: Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie
    Article Snippet: Universal primers (forward, 5′-AGAGTTTGATCATGGCTCAG-3′; and reverse, 5′-GGTTACCTTGTTACGACTT-3′) were used to PCR amplify a 1,465-bp 16S rRNA gene segment, using GoTaq Green master mix (Promega, Madison, WI). .. The amplicon was subsequently double digested with AluI and MboI (New England BioLabs, Ipswich, MA) at 37°C overnight and electrophoresed in a 4% MetaPhor agarose gel (Lonza, Rockland, ME) as previously described ( ). .. Strains of A. hydrophila (ATCC 7966), A. caviae (ATCC 15468), and A. veronii (ATCC 9071) were used as positive controls.

    Ethanol Precipitation:

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Sodium azide was added to a final concentration of 0.2% (w/v), and cells were collected by centrifugation (5,000 g , 10 min at 4°C). .. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. DNA concentration was determined spectrophotometrically and samples were stored at −80°C.

    Article Title: Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation
    Article Snippet: Isolated nuclei from 1 × 107 cells were incubated overnight at 37 °C in 100 μl of AluI buffer (New England Biolabs; 50 m m NaCl, 10 m m Tris-Cl, 10 m m MgCl2 , 1 m m DTT, pH 7.9) with 1 unit/μl of AluI. .. Following the addition of 100 μl of 2× proteinase K digestion buffer (100 m m TrisCl, 200 m m NaCl, 2 m m Na2 EDTA, 1% SDS, pH 7.5) for 2 h at 55 °C, 100 μl of AluI buffer plus 100 μl of 2× proteinase K buffer containing 100 μg of proteinase K were added and incubated overnight at 37 °C.

    Concentration Assay:

    Article Title: Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication
    Article Snippet: Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ). .. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), digested with Alu I (New England Biolabs, Beverly, MA, USA) and purified by phenol:chloroform extraction followed by ethanol precipitation ( ).

    BAC Assay:

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: BAC clones were cultured and DNA was purified using QIAGEN Plasmid Midi Kit (Qiagen 12143, Hilden, Germany). .. Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). .. After incubating for 2 h at 37 °C, 5 mM EDTA was added to terminate the reaction.

    Lysis:

    Article Title: Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie
    Article Snippet: Briefly, bacterial DNA was obtained through lysis using BR-A buffer (GenScript, Piscataway, NJ). .. The amplicon was subsequently double digested with AluI and MboI (New England BioLabs, Ipswich, MA) at 37°C overnight and electrophoresed in a 4% MetaPhor agarose gel (Lonza, Rockland, ME) as previously described ( ).

    Article Title: ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner
    Article Snippet: Cells were resuspended in proteinase K lysis buffer (10 mM Tris-HCl, pH 8; 100 mM EDTA; 0.5% SDS; 20 μg/mL RNase A; 100 μg/mL proteinase K) and incubated at 37°C with agitation for five hours. .. Two equal volume phenol:chloroform:isoamyl alcohol (25:24:1, pH 8) extractions were performed, followed by two ethanol precipitations and final resuspension in H2 O. DNA was digested overnight with AluI and MboI (New England Biolabs).

    Fluorescence In Situ Hybridization:

    Article Title: Condensins are Required for Maintenance of Nuclear Architecture
    Article Snippet: Paragraph title: 2.4. FISH Probe Preparation ... Purified BAC DNA was amplified using Whole genome amplification kit (Sigma-Aldrich WGA1), 20 µg of amplified DNA was then digested using a cocktail of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs, Ipswich, MA, USA) overnight at 37 °C, ethanol precipitated, and resuspended in 36 µL ddH2 O. DNA was denatured at 100 °C for 1 min, and then 3'-end-labeled with unmodified aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany).

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  • 99
    New England Biolabs t4 dna ligase buffer
    Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat  = 2.2 ± 0.2 × 10 −4  s −1  and K M  = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0  was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat  is 8 × 10 −3  s −1  with an upper threshold for the K M  estimated to be 1 nM.
    T4 Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs rapid pngase f buffer
    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in  Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.
    Rapid Pngase F Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs buffer 4
    Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42,  32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t  or S b ) and cleaved strands (P 16  or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1  and 0.3 min −1  for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1  and 0.5 min −1  for top and bottom strands, respectively.
    Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat  = 2.2 ± 0.2 × 10 −4  s −1  and K M  = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0  was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat  is 8 × 10 −3  s −1  with an upper threshold for the K M  estimated to be 1 nM.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat = 2.2 ± 0.2 × 10 −4 s −1 and K M = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0 was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat is 8 × 10 −3 s −1 with an upper threshold for the K M estimated to be 1 nM.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Ligation, Concentration Assay

    Ligation of DNA splinted by RNA. (Left) Outline of the ligation assay: a 5′-phosphorylated, 3′-FAM labelled DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor’ oligonucleotide are annealed to a complementary RNA or DNA splint. This substrate was reacted with a ligase to form a mixture of unreacted starting material, adenylylated DNA and ligated product. The products were denatured, separated on CE, and detected by fluorescence. (Right) ligation of the standard RNA-splinted substrate in ligase assay buffer for 15 min at 25°C with ( A ) no enzyme, ( B ) 1 µM T4 DNA ligase and 10 µM ATP, ( C ) 1 µM T4 DNA ligase and 1 mM ATP, ( D ) 100 nM PBCV-1 DNA ligase and 10 µM ATP, and ( E ) 100 nM PBCV-1 DNA ligase and 1 mM ATP. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by coelution with synthetically prepared standards.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Ligation of DNA splinted by RNA. (Left) Outline of the ligation assay: a 5′-phosphorylated, 3′-FAM labelled DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor’ oligonucleotide are annealed to a complementary RNA or DNA splint. This substrate was reacted with a ligase to form a mixture of unreacted starting material, adenylylated DNA and ligated product. The products were denatured, separated on CE, and detected by fluorescence. (Right) ligation of the standard RNA-splinted substrate in ligase assay buffer for 15 min at 25°C with ( A ) no enzyme, ( B ) 1 µM T4 DNA ligase and 10 µM ATP, ( C ) 1 µM T4 DNA ligase and 1 mM ATP, ( D ) 100 nM PBCV-1 DNA ligase and 10 µM ATP, and ( E ) 100 nM PBCV-1 DNA ligase and 1 mM ATP. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by coelution with synthetically prepared standards.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Ligation, Fluorescence

    Detection of defined amounts of luciferase mRNA from a mixture with Jurkat total RNA through ligation of specific DNA probes (Probe set A) and detection by qPCR. RNA/DNA probe mixtures were annealed then incubated with either T4 DNA ligase (open circle) or PBCV-1 DNA ligase (×). The qPCR C q  for each experiment was recorded, with lower C q  indicating higher concentration of ligated probes. ( A ) Ligation time course with either 1 (solid line) or 0.01 ng (dotted line) of luciferase mRNA target, 0–8 h ligation time. ( B ) Dependence of C q  after probe ligation with target luciferase mRNA present over a 7 log concentration range using a 2 h ligation time. NTC = no template control. The error bars show one standard deviation of the average of a minimum of three replicates.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Detection of defined amounts of luciferase mRNA from a mixture with Jurkat total RNA through ligation of specific DNA probes (Probe set A) and detection by qPCR. RNA/DNA probe mixtures were annealed then incubated with either T4 DNA ligase (open circle) or PBCV-1 DNA ligase (×). The qPCR C q for each experiment was recorded, with lower C q indicating higher concentration of ligated probes. ( A ) Ligation time course with either 1 (solid line) or 0.01 ng (dotted line) of luciferase mRNA target, 0–8 h ligation time. ( B ) Dependence of C q after probe ligation with target luciferase mRNA present over a 7 log concentration range using a 2 h ligation time. NTC = no template control. The error bars show one standard deviation of the average of a minimum of three replicates.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Luciferase, Ligation, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay, Standard Deviation

    Ligation of RNA-splinted DNA substrates by PBCV-1 and T4 DNA ligases under general reaction conditions. The 16 RNA-splinted DNA substrates, representing all possible base pairs at the ligation junction, were reacted and the extent of ligation and abortive adenylylation measured. The bases listed on the X axis (dN/pdN) refer to the identity of the base of the DNA acceptor at the ligation junction (dN) and the identity of the phosphorylated base on the donor at the ligation junction (pdN). For all substrates, the correct Watson–Crick base-pairing partner was present in the RNA splint. All reactions were incubated at 37°C in 50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 nM RNA-splinted DNA substrate and ( A ) 100 nM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( B ) 100 nM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( C ) 1 µM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( D ) 1 µM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( E ) 1 µM T4 DNA ligase and 10 µM ATP for 15 min; and ( F ) 1 µM T4 DNA ligase and 10 µM ATP for 4 h. Here the total height of the bar indicates the fraction of starting material converted to products, with the solid portion indicating ligation product yield and the hashed portion indicating AppDNA yield.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Ligation of RNA-splinted DNA substrates by PBCV-1 and T4 DNA ligases under general reaction conditions. The 16 RNA-splinted DNA substrates, representing all possible base pairs at the ligation junction, were reacted and the extent of ligation and abortive adenylylation measured. The bases listed on the X axis (dN/pdN) refer to the identity of the base of the DNA acceptor at the ligation junction (dN) and the identity of the phosphorylated base on the donor at the ligation junction (pdN). For all substrates, the correct Watson–Crick base-pairing partner was present in the RNA splint. All reactions were incubated at 37°C in 50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 nM RNA-splinted DNA substrate and ( A ) 100 nM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( B ) 100 nM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( C ) 1 µM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( D ) 1 µM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( E ) 1 µM T4 DNA ligase and 10 µM ATP for 15 min; and ( F ) 1 µM T4 DNA ligase and 10 µM ATP for 4 h. Here the total height of the bar indicates the fraction of starting material converted to products, with the solid portion indicating ligation product yield and the hashed portion indicating AppDNA yield.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Ligation, Dominant Negative Mutation, Incubation

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in  Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Binding Assay, Sequencing, Incubation, SDS Page

    N-glycopeptide enrichment from human serum tryptic peptides by Fbs1 GYR. ( a ) Left panel: The total ion chromatogram (TIC) from an LC-MS analysis of HSA-depleted human serum tryptic peptides without Fbs1 enrichment (pre-enrichment) or the peptides from Fbs1 GYR enrichment. Right panel: TIC from an LC-MS analysis of peptides enriched by Fbs1 GYR treated with active PNGase F (blue curve) or heat-inactivated PNGase F (95 °C, 10 min) (orange curve). A representative TIC profile is shown from three experiments. ( b ) N-glycan profiling of human serum (-HSA) tryptic peptides without Fbs1 enrichment (pre-enrichment, dark curve) or the peptides from Fbs1 GYR Enrichment (red curve) by 2-AB labelling and UPLC. Glycan structures are assigned to major peaks according to the glucose unit of each peak ( Supplementary Fig. 8 ). The reduced peaks in the Fbs1 GYR enrichment sample are labelled with an ‘*' The red curve is offset for better comparison with the black curve. A representative glycan profile is shown from two experiments. ( c ) N-glycosite identification using PNGase F deglycosylation in  18 O water. HSA-depleted human serum tryptic peptides without Fbs1 enrichment (pre-enrichment) or enriched by Fbs1 GYR (Fbs1 GYR enrichment) were deglycosylated by PNGase F in  18 O water. Upon N-glycan removal, asparagine (N) in the N-glycosite is deamidated to aspartic acid resulting in a peptide tagged with an additional 2.988 daltons. Peptide spectra with N[+2.988] within the canonical N-glycosylation motif N-X-T/S can be confidently assigned as N-glycopeptides. Detailed MS spectrum information is in  Supplementary Data 1 and 2 . The MS data were combined from two mass spectrometric experiments. ( d ) Comparison of unique N-glycosites and N-glycoproteins identified without (pre-enrichment) or with Fbs1 GYR enrichment. Left panel: 183 and 477 unique N-glycosites were identified in pre-enrichment and Fbs1 GYR enrichment, respectively. One hundred and seventy-two (94%) of the 183 N-glycosites in the pre-enrichment were also identified in the Fbs1 GYR enrichment sample. Right panel: 89 and 230 N-glycoproteins were identified in pre-enrichment and Fbs1 GYR enrichment, respectively. Eighty-three (93%) of the 89 N-glycoprotein in the pre-enrichment were also identified in the Fbs1 GYR enrichment sample. Detailed N-glycosite and N-glycoprotein information is in  Supplementary Data 1 and 2 .

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: N-glycopeptide enrichment from human serum tryptic peptides by Fbs1 GYR. ( a ) Left panel: The total ion chromatogram (TIC) from an LC-MS analysis of HSA-depleted human serum tryptic peptides without Fbs1 enrichment (pre-enrichment) or the peptides from Fbs1 GYR enrichment. Right panel: TIC from an LC-MS analysis of peptides enriched by Fbs1 GYR treated with active PNGase F (blue curve) or heat-inactivated PNGase F (95 °C, 10 min) (orange curve). A representative TIC profile is shown from three experiments. ( b ) N-glycan profiling of human serum (-HSA) tryptic peptides without Fbs1 enrichment (pre-enrichment, dark curve) or the peptides from Fbs1 GYR Enrichment (red curve) by 2-AB labelling and UPLC. Glycan structures are assigned to major peaks according to the glucose unit of each peak ( Supplementary Fig. 8 ). The reduced peaks in the Fbs1 GYR enrichment sample are labelled with an ‘*' The red curve is offset for better comparison with the black curve. A representative glycan profile is shown from two experiments. ( c ) N-glycosite identification using PNGase F deglycosylation in 18 O water. HSA-depleted human serum tryptic peptides without Fbs1 enrichment (pre-enrichment) or enriched by Fbs1 GYR (Fbs1 GYR enrichment) were deglycosylated by PNGase F in 18 O water. Upon N-glycan removal, asparagine (N) in the N-glycosite is deamidated to aspartic acid resulting in a peptide tagged with an additional 2.988 daltons. Peptide spectra with N[+2.988] within the canonical N-glycosylation motif N-X-T/S can be confidently assigned as N-glycopeptides. Detailed MS spectrum information is in Supplementary Data 1 and 2 . The MS data were combined from two mass spectrometric experiments. ( d ) Comparison of unique N-glycosites and N-glycoproteins identified without (pre-enrichment) or with Fbs1 GYR enrichment. Left panel: 183 and 477 unique N-glycosites were identified in pre-enrichment and Fbs1 GYR enrichment, respectively. One hundred and seventy-two (94%) of the 183 N-glycosites in the pre-enrichment were also identified in the Fbs1 GYR enrichment sample. Right panel: 89 and 230 N-glycoproteins were identified in pre-enrichment and Fbs1 GYR enrichment, respectively. Eighty-three (93%) of the 89 N-glycoprotein in the pre-enrichment were also identified in the Fbs1 GYR enrichment sample. Detailed N-glycosite and N-glycoprotein information is in Supplementary Data 1 and 2 .

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Liquid Chromatography, Mass Spectrometry

    Fbs1 binds to diverse types of N-glycomolecules. ( a ) Fbs1 binding to RNase B is N-glycan dependent. RNase B or deglycosylated RNase B was subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Lane 1, RNase B input control (CTL). Lane 2, essentially no RNase B deglycosylated by PNGase F is pulled down by Fbs1. Lane 3, RNase B with N-glycans is efficiently pulled down by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 binds to the N-glycosylated heavy chain of human IgG. Denatured and reduced human IgG were subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Heavy chains of human IgGs are typically N-glycosylated. Lane 1 is a control showing the IgG light chain and heavy chain. Lane 2 is Fbs1 beads only. Some SNAP-Fbs1 protein leaches from the prototype Fbs1 beads (denoted by an asterisk). Lanes 3 and 4 show that only the glycosylated heavy chain is bound by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( c ) Fbs1 binding affinity (Kd value) to sialylglycopeptide (SGP), M3N2 and M3N2F was measured by isothermal titration calorimetry. Structures of SGP, M3N2 and M3N2F are shown in the left panel. M3N2F is M3N2 with α1-6 fucosylation at the reducing end GlcNAc. The left panel summarizes the Kd values of SGP ( n =4), M3N2 ( n =5) and M3N2F ( n =5) interacting with wt Fbs1. There is no significant difference between the Kd values of M3N2 and M3N2F ( P  value 0.85 > 0.05, mean±s.e.m.,  t -test, two-tailed). ( d ) wt Fbs1 shows binding bias to different N-glycopeptides. SGP was labelled with TMR fluorophore to facilitate detection. N-glycans of SGP-TMR (1) were then trimmed with exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Binding of the trimmed glycopeptides to Fbs1 beads was analysed. The relative binding affinity to wt Fbs1 is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is only indicated in N-glycopeptide structure 1. Results represent the mean±s.e.m. of three replicates.

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: Fbs1 binds to diverse types of N-glycomolecules. ( a ) Fbs1 binding to RNase B is N-glycan dependent. RNase B or deglycosylated RNase B was subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Lane 1, RNase B input control (CTL). Lane 2, essentially no RNase B deglycosylated by PNGase F is pulled down by Fbs1. Lane 3, RNase B with N-glycans is efficiently pulled down by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 binds to the N-glycosylated heavy chain of human IgG. Denatured and reduced human IgG were subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Heavy chains of human IgGs are typically N-glycosylated. Lane 1 is a control showing the IgG light chain and heavy chain. Lane 2 is Fbs1 beads only. Some SNAP-Fbs1 protein leaches from the prototype Fbs1 beads (denoted by an asterisk). Lanes 3 and 4 show that only the glycosylated heavy chain is bound by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( c ) Fbs1 binding affinity (Kd value) to sialylglycopeptide (SGP), M3N2 and M3N2F was measured by isothermal titration calorimetry. Structures of SGP, M3N2 and M3N2F are shown in the left panel. M3N2F is M3N2 with α1-6 fucosylation at the reducing end GlcNAc. The left panel summarizes the Kd values of SGP ( n =4), M3N2 ( n =5) and M3N2F ( n =5) interacting with wt Fbs1. There is no significant difference between the Kd values of M3N2 and M3N2F ( P value 0.85 > 0.05, mean±s.e.m., t -test, two-tailed). ( d ) wt Fbs1 shows binding bias to different N-glycopeptides. SGP was labelled with TMR fluorophore to facilitate detection. N-glycans of SGP-TMR (1) were then trimmed with exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Binding of the trimmed glycopeptides to Fbs1 beads was analysed. The relative binding affinity to wt Fbs1 is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is only indicated in N-glycopeptide structure 1. Results represent the mean±s.e.m. of three replicates.

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Binding Assay, SDS Page, CTL Assay, Isothermal Titration Calorimetry, Two Tailed Test, Fluorescence

    Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42,  32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t  or S b ) and cleaved strands (P 16  or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1  and 0.3 min −1  for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1  and 0.5 min −1  for top and bottom strands, respectively.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42, 32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t or S b ) and cleaved strands (P 16 or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1 and 0.3 min −1 for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1 and 0.5 min −1 for top and bottom strands, respectively.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Selection, Polyacrylamide Gel Electrophoresis

    Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM  3 H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. ( A ) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. ( B ) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM 3 H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. ( A ) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. ( B ) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Concentration Assay

    Strand-specific nicking. ( A ) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was  32 P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S t  and S b , from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P 16  from the top strand, P 12  from the bottom). ( B ) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min −1 . Data points for the bottom strand are connected by a dashed line.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Strand-specific nicking. ( A ) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was 32 P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S t and S b , from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P 16 from the top strand, P 12 from the bottom). ( B ) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min −1 . Data points for the bottom strand are connected by a dashed line.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Polyacrylamide Gel Electrophoresis

    Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. ( A ) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. ( B ) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. ( A ) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. ( B ) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Plasmid Preparation, Electrophoresis, Concentration Assay