bamhi  (New England Biolabs)


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  • 99
    Name:
    BamHI HF
    Description:
    BamHI HF 50 000 units
    Catalog Number:
    R3136L
    Price:
    244
    Size:
    50 000 units
    Category:
    Restriction Enzymes
    Score:
    85
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    New England Biolabs bamhi
    BamHI HF
    BamHI HF 50 000 units
    https://www.bioz.com/result/bamhi/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bamhi - by Bioz Stars, 2019-10
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    Images

    1) Product Images from "Plasmid-Mediated High-Level Gentamicin Resistance among Enteric Bacteria Isolated from Pet Turtles in Louisiana"

    Article Title: Plasmid-Mediated High-Level Gentamicin Resistance among Enteric Bacteria Isolated from Pet Turtles in Louisiana

    Journal:

    doi: 10.1128/AEM.72.1.306-312.2006

    HindIII and BamHI digestion profiles of R plasmids pSal5 (lanes 1 and 4), pAri40 (lanes 2 and 5), and pEnt19 (lanes 3 and 6). A λ-DNA-HindIII digest, used as a molecular standard, is indicated on the left. Arrowheads indicate bands carrying the
    Figure Legend Snippet: HindIII and BamHI digestion profiles of R plasmids pSal5 (lanes 1 and 4), pAri40 (lanes 2 and 5), and pEnt19 (lanes 3 and 6). A λ-DNA-HindIII digest, used as a molecular standard, is indicated on the left. Arrowheads indicate bands carrying the

    Techniques Used:

    2) Product Images from "Processes of copy-number change in human DNA: The dynamics of ?-globin gene deletion"

    Article Title: Processes of copy-number change in human DNA: The dynamics of ?-globin gene deletion

    Journal:

    doi: 10.1073/pnas.0602690103

    Detection of de novo deletions in the α-globin gene region. ( A ) The region analyzed, showing the α-globin genes and pseudogene, plus BamHI cleavage sites used for size fractionation of genomic DNA and nested PCR primers (arrows) used to
    Figure Legend Snippet: Detection of de novo deletions in the α-globin gene region. ( A ) The region analyzed, showing the α-globin genes and pseudogene, plus BamHI cleavage sites used for size fractionation of genomic DNA and nested PCR primers (arrows) used to

    Techniques Used: Fractionation, Nested PCR

    3) Product Images from "Novel N4-Like Bacteriophages of Pectobacterium atrosepticum"

    Article Title: Novel N4-Like Bacteriophages of Pectobacterium atrosepticum

    Journal: Pharmaceuticals

    doi: 10.3390/ph11020045

    Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.
    Figure Legend Snippet: Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.

    Techniques Used: Marker, Concentration Assay

    4) Product Images from "Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection"

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114208

    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).
    Figure Legend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation

    5) Product Images from "The Effects of Apolipoprotein F Deficiency on High Density Lipoprotein Cholesterol Metabolism in Mice"

    Article Title: The Effects of Apolipoprotein F Deficiency on High Density Lipoprotein Cholesterol Metabolism in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031616

    Generation of Apolipoprotein F Deficient Mice. A. Schematic diagram (not to scale) showing the wild type and deleted ApoF alleles. Features are depicted as follows: Exons- white arrows, Beta galactosidase reporter gene- “LacZ” black arrow, PGK promoter- grey arrow, Neomycin resistance gene- “Neo” black arrow, BamHI restriction sites- vertical lines, and the location of Southern Blotting probe- asterisk. B. Southern blot confirming successful targeting and genomic location of null allele: WT allele yields a 3.9 kb band, while the Null allele is 2.4 kb. The left panel contains ES cell DNA, while the right panel depicts livers from the mice. C. Real Time RT-PCR data on ApoF mRNA in the livers of female wild type (+/+), heterozygous (+/−), and homozygous (−/−) ApoF deficient mice.
    Figure Legend Snippet: Generation of Apolipoprotein F Deficient Mice. A. Schematic diagram (not to scale) showing the wild type and deleted ApoF alleles. Features are depicted as follows: Exons- white arrows, Beta galactosidase reporter gene- “LacZ” black arrow, PGK promoter- grey arrow, Neomycin resistance gene- “Neo” black arrow, BamHI restriction sites- vertical lines, and the location of Southern Blotting probe- asterisk. B. Southern blot confirming successful targeting and genomic location of null allele: WT allele yields a 3.9 kb band, while the Null allele is 2.4 kb. The left panel contains ES cell DNA, while the right panel depicts livers from the mice. C. Real Time RT-PCR data on ApoF mRNA in the livers of female wild type (+/+), heterozygous (+/−), and homozygous (−/−) ApoF deficient mice.

    Techniques Used: Mouse Assay, Southern Blot, Quantitative RT-PCR

    6) Product Images from "Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena"

    Article Title: Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

    Journal: Genes

    doi: 10.3390/genes9040179

    Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.
    Figure Legend Snippet: Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.

    Techniques Used: Mutagenesis, Plasmid Preparation, Homologous Recombination, Southern Blot, Expressing, Molecular Weight, Western Blot

    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).
    Figure Legend Snippet: Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Techniques Used: Knock-Out, Plasmid Preparation, Homologous Recombination, Southern Blot, Molecular Weight

    7) Product Images from "Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli"

    Article Title: Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

    Journal:

    doi: 10.1016/j.pep.2012.05.007

    (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The BamHI and SalI restriction sites used to clone TH are unique in the vector
    Figure Legend Snippet: (A) hTH/pMALc2H 10 T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The BamHI and SalI restriction sites used to clone TH are unique in the vector

    Techniques Used: Construct, Recombinant, Sequencing, Plasmid Preparation

    8) Product Images from "Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries"

    Article Title: Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064131

    Regeneration of G 2 N 5 population from PCR products. (A) Sequence of the PCR product. PstI and BamHI restriction sites are shown in boxes. Restriction enzyme cleavage sites are represented by arrows. (B) Digestion of amplified DGR36 sequence with BamHI and PstI individually (lanes 2 and 3) and in combination (lane 4). Coloured arrows indicate fragments comprised of sequences of the same colour in Panel A. Fragments consisting of only the first fifteen nucleotides of the 5′ regions of the PCR products in the single enzyme digestion experiments cannot be seen as only polymerized segments acquire 32 P during amplification.
    Figure Legend Snippet: Regeneration of G 2 N 5 population from PCR products. (A) Sequence of the PCR product. PstI and BamHI restriction sites are shown in boxes. Restriction enzyme cleavage sites are represented by arrows. (B) Digestion of amplified DGR36 sequence with BamHI and PstI individually (lanes 2 and 3) and in combination (lane 4). Coloured arrows indicate fragments comprised of sequences of the same colour in Panel A. Fragments consisting of only the first fifteen nucleotides of the 5′ regions of the PCR products in the single enzyme digestion experiments cannot be seen as only polymerized segments acquire 32 P during amplification.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification

    9) Product Images from "Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins"

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

    Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).
    Figure Legend Snippet: Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

    Techniques Used: Electrophoresis, Sequencing

    10) Product Images from "DNA slip-outs cause RNA polymerase II arrest in vitro: potential implications for genetic instability"

    Article Title: DNA slip-outs cause RNA polymerase II arrest in vitro: potential implications for genetic instability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr429

    ( A ) Preparation of DNA substrates. The plasmid was cut with restriction enzymes EcoRI and BamHI to yield a fragment containing the T7 RNAP and RNAPII promoters. Oligonucleotides were designed such that, when annealed, they would produce a slip-out and contain BamHI and SalI sticky ends. The olgonucleotides were ligated to the promoter fragment through the BamHI site. Oligonucleotides were not 5′ phosphorylated in order to prevent self-ligation and were used in excess to promote ligation of most promoter fragments to an oligonucleotide. A minor fraction, consisting of promoter fragments ligated through the BamHI site still appeared, however, this fraction does not interfere with our analysis because its transcription product is longer than that produced by our construct of interest. Due to lack of 5′ phosphorylation of oligonucleotides, our substrates contain a nick in the NTS, however, the presence of a nick on the NTS had no effect on the observed transcription arrest ( Supplementary Figure SI6 ). Ligation was followed by EcoRI digestion to obtain the final substrates containing one promoter fragment, with T7 RNAP and RNAPII promoters (designated by the arrow), and a slip-out. ( B ) Sequences used in this study. Oligonucleotides sequences, which will become the TS or the NTS when ligated to a promoter fragment, are shown. Different combinations of oligonucleotides result in different inserts in the middle but have the same flanking sequence (shown on top). The sequence of different inserts is indicated in the 5′ to 3′ direction. NI (‘No insert’) refers to oligonucleotides containing only the flanking sequence. The designation of inserts is as follows: CAG, 20 CAG repeats; CTG, 20 CTG repeats; R1, random sequence; R1C, complementary to R1; R2, random sequence; R2C, complementary to R2; H1, capable of forming a perfect hairpin; H2, capable of forming a perfect hairpin.
    Figure Legend Snippet: ( A ) Preparation of DNA substrates. The plasmid was cut with restriction enzymes EcoRI and BamHI to yield a fragment containing the T7 RNAP and RNAPII promoters. Oligonucleotides were designed such that, when annealed, they would produce a slip-out and contain BamHI and SalI sticky ends. The olgonucleotides were ligated to the promoter fragment through the BamHI site. Oligonucleotides were not 5′ phosphorylated in order to prevent self-ligation and were used in excess to promote ligation of most promoter fragments to an oligonucleotide. A minor fraction, consisting of promoter fragments ligated through the BamHI site still appeared, however, this fraction does not interfere with our analysis because its transcription product is longer than that produced by our construct of interest. Due to lack of 5′ phosphorylation of oligonucleotides, our substrates contain a nick in the NTS, however, the presence of a nick on the NTS had no effect on the observed transcription arrest ( Supplementary Figure SI6 ). Ligation was followed by EcoRI digestion to obtain the final substrates containing one promoter fragment, with T7 RNAP and RNAPII promoters (designated by the arrow), and a slip-out. ( B ) Sequences used in this study. Oligonucleotides sequences, which will become the TS or the NTS when ligated to a promoter fragment, are shown. Different combinations of oligonucleotides result in different inserts in the middle but have the same flanking sequence (shown on top). The sequence of different inserts is indicated in the 5′ to 3′ direction. NI (‘No insert’) refers to oligonucleotides containing only the flanking sequence. The designation of inserts is as follows: CAG, 20 CAG repeats; CTG, 20 CTG repeats; R1, random sequence; R1C, complementary to R1; R2, random sequence; R2C, complementary to R2; H1, capable of forming a perfect hairpin; H2, capable of forming a perfect hairpin.

    Techniques Used: Plasmid Preparation, Ligation, Produced, Construct, Sequencing, CTG Assay

    11) Product Images from "Evidence that Plasmid-Borne Botulinum Neurotoxin Type B Genes Are Widespread among Clostridium botulinum Serotype B Strains"

    Article Title: Evidence that Plasmid-Borne Botulinum Neurotoxin Type B Genes Are Widespread among Clostridium botulinum Serotype B Strains

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004829

    BamHI, HindIII, SacI and EcoRV restrictions of the bont /B PCR products obtained from strains CDC-1758 (lanes 1, 5, 9, 14); CDC-1828 (lanes 2, 6, 10, 15); CDC-1436 (lanes 3, 7, 11, 16); CDC-4848 (lanes 4, 8, 12); CDC-816 (lane 13). M.S. (Molecular standard, 1 kb Promega).
    Figure Legend Snippet: BamHI, HindIII, SacI and EcoRV restrictions of the bont /B PCR products obtained from strains CDC-1758 (lanes 1, 5, 9, 14); CDC-1828 (lanes 2, 6, 10, 15); CDC-1436 (lanes 3, 7, 11, 16); CDC-4848 (lanes 4, 8, 12); CDC-816 (lane 13). M.S. (Molecular standard, 1 kb Promega).

    Techniques Used: Polymerase Chain Reaction

    12) Product Images from "A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections"

    Article Title: A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

    Journal: RNA

    doi: 10.1261/rna.576908

    Analysis of BamHI restriction digestion of biotinylated RT-PCR product off of streptavidin resin. Lanes: ( A ) biotinylated RT primer; ( B ) biotinylated PCR primer; ( C ) crude RT-PCR reaction; ( D ) crude in solution restriction digestion with BamHI; ( E ) DNA eluted from resin-supported restriction digestion with BamHI.
    Figure Legend Snippet: Analysis of BamHI restriction digestion of biotinylated RT-PCR product off of streptavidin resin. Lanes: ( A ) biotinylated RT primer; ( B ) biotinylated PCR primer; ( C ) crude RT-PCR reaction; ( D ) crude in solution restriction digestion with BamHI; ( E ) DNA eluted from resin-supported restriction digestion with BamHI.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    13) Product Images from "Novel N4-Like Bacteriophages of Pectobacterium atrosepticum"

    Article Title: Novel N4-Like Bacteriophages of Pectobacterium atrosepticum

    Journal: Pharmaceuticals

    doi: 10.3390/ph11020045

    Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.
    Figure Legend Snippet: Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.

    Techniques Used: Marker, Concentration Assay

    14) Product Images from "Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility"

    Article Title: Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility

    Journal: Scientific Reports

    doi: 10.1038/srep33186

    CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI. ( a ) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary 46 . ( b ) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. ( c ) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.
    Figure Legend Snippet: CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI. ( a ) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary 46 . ( b ) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. ( c ) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.

    Techniques Used: Construct, Sequencing, Imaging, Software, Agarose Gel Electrophoresis, Standard Deviation

    15) Product Images from "Isolation and characterisation of a ruminant alphaherpesvirus closely related to bovine herpesvirus 1 in a free-ranging red deer"

    Article Title: Isolation and characterisation of a ruminant alphaherpesvirus closely related to bovine herpesvirus 1 in a free-ranging red deer

    Journal: BMC Veterinary Research

    doi: 10.1186/1746-6148-3-26

    BamHI (a) and BstEII (b) restriction endonuclease profiles of the Anlier isolate and ruminant alphaherpesviruses.
    Figure Legend Snippet: BamHI (a) and BstEII (b) restriction endonuclease profiles of the Anlier isolate and ruminant alphaherpesviruses.

    Techniques Used:

    16) Product Images from "Phenotypic and Molecular Analysis of Tellurite Resistance among Enterohemorrhagic Escherichia coli O157:H7 and Sorbitol-Fermenting O157:NM Clinical Isolates"

    Article Title: Phenotypic and Molecular Analysis of Tellurite Resistance among Enterohemorrhagic Escherichia coli O157:H7 and Sorbitol-Fermenting O157:NM Clinical Isolates

    Journal:

    doi: 10.1128/JCM.43.1.452-454.2005

    Hybridization of BamHI-PstI-digested genomic DNA from EHEC O157 strains and controls with the terC probe. M, molecular weight marker (1-kb DNA ladder; Gibco-BRL). In lanes 1 to 7, the following strains are displayed (serotype, Te-MIC in micrograms per
    Figure Legend Snippet: Hybridization of BamHI-PstI-digested genomic DNA from EHEC O157 strains and controls with the terC probe. M, molecular weight marker (1-kb DNA ladder; Gibco-BRL). In lanes 1 to 7, the following strains are displayed (serotype, Te-MIC in micrograms per

    Techniques Used: Hybridization, Molecular Weight, Marker

    17) Product Images from "Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq"

    Article Title: Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq

    Journal: mBio

    doi: 10.1128/mBio.00315-10

    Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a DNA fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, BamHI, which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).
    Figure Legend Snippet: Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a DNA fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, BamHI, which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).

    Techniques Used: Sequencing, Mutagenesis, Selection, Ligation, Amplification, Real-time Polymerase Chain Reaction, Flow Cytometry

    18) Product Images from "Design and Characterization of Bioengineered Cancer-Like Stem Cells"

    Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141172

    Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
    Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Techniques Used: Subcloning, Clone Assay, DNA Sequencing

    Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
    Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Techniques Used: Subcloning, Clone Assay, DNA Sequencing

    19) Product Images from "TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression"

    Article Title: TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186568

    pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.
    Figure Legend Snippet: pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.

    Techniques Used: Positron Emission Tomography

    Screening for E . coli colonies transformed with recombinant pET-BccI. Plasmid preparations from cultures inoculated with transformed colonies were digested with BamHI and subjected to electrophoresis. A ) For BRP recombinants pET-BccI screening, the presence of a 283 bp DNA band indicated there was no recombination (A3, A7, A9), whereas the presence of a 636 bp insert DNA band showed a successful recombination (A2, A4, A5, A6, A8). B ) For CAT recombinants pET-BccI screening, again the presence of a 283 bp DNA band indicated there was no recombination (B2, B5, B6, B8), whereas the presence of a 924 bp insert DNA band showed a successful recombination (B3, B4, B7, B9).
    Figure Legend Snippet: Screening for E . coli colonies transformed with recombinant pET-BccI. Plasmid preparations from cultures inoculated with transformed colonies were digested with BamHI and subjected to electrophoresis. A ) For BRP recombinants pET-BccI screening, the presence of a 283 bp DNA band indicated there was no recombination (A3, A7, A9), whereas the presence of a 636 bp insert DNA band showed a successful recombination (A2, A4, A5, A6, A8). B ) For CAT recombinants pET-BccI screening, again the presence of a 283 bp DNA band indicated there was no recombination (B2, B5, B6, B8), whereas the presence of a 924 bp insert DNA band showed a successful recombination (B3, B4, B7, B9).

    Techniques Used: Transformation Assay, Recombinant, Positron Emission Tomography, Plasmid Preparation, Electrophoresis

    The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.
    Figure Legend Snippet: The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.

    Techniques Used: Expressing, Plasmid Preparation, Positron Emission Tomography, Derivative Assay, Selection, Transformation Assay, Clone Assay, Recombinant, Ligation

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    Centrifugation:

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
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    Amplification:

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    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
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    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
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    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
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    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
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    Synthesized:

    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
    Article Snippet: Plasmid DNA standards were synthesized for CowM3 and Rum-2-bac assays by amplifying a segment of the hydrolase domain (HD) superfamily and 16S rRNA loci, respectively, using PCR ( ). .. Plasmids were extracted using a PureLink Quick plasmid miniprep kit (Life Technologies) and then linearized with BamHI-HF enzyme (New England BioLabs, Ipswich, MA).

    Construct:

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    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
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    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
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    Real-time Polymerase Chain Reaction:

    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
    Article Snippet: American Type Culture Collection (ATCC) bacterial strains were used to prepare qPCR standard curves for E. coli (ATCC 25922), enterococci ( Enterococcus faecalis ATCC 29212), and GenBac ( Bacteroides thetaiotaomicron ATCC 29741). .. Plasmids were extracted using a PureLink Quick plasmid miniprep kit (Life Technologies) and then linearized with BamHI-HF enzyme (New England BioLabs, Ipswich, MA).

    Modification:

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
    Article Snippet: All coding regions were codon-optimized for E. coli expression. .. DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site. .. The resulting DNA constructs (verified by sequencing) were transformed into E. coli strain BL21 C41(DE3).

    Incubation:

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    Article Snippet: Digest reactions were incubated at 37 °C for 1 h and were then inactivated by incubation at 75 °C for 30 min. Fractions were analyzed by field-inversion gel electrophoresis, and fractions containing approximately 40 kb pieces of DNA were utilized in cosmid construction. .. These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase. .. Cut vector was purified with a GeneJET PCR Purification Kit (Thermo Scientific).

    Gel Extraction:

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: The complete fur gene ( 411 base pairs) was then amplified by standard PCR methods using Pfu polymerase and the primers 5′-TACTTATG CC GCGGCCGC TCAGGATTTGAGGGGTTTGA-3′and 5′-CACCTTCTAA GGATCC ATGTCATCCGA AAACACCGA-3′(restriction sites underlined). .. After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. The resultant Mb-Fur pHIS8 plasmid encodes for amino acids 1 to 411 of M. algicola Fur with an amino-terminal histidine tag and a thrombin cleavage site.

    Cell Culture:

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. Expression of recombinant Mb-Fur was carried out in BL21 E. coli cells (Stratagene).

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
    Article Snippet: DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site. .. The resulting DNA constructs (verified by sequencing) were transformed into E. coli strain BL21 C41(DE3).

    Expressing:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: Abdurin libraries were subcloned in frame with pIII into pIFF6, a phagemid expression vector containing the pIII gene sequence of filamentous phage f1 for the expression of recombinant protein inserts at the amino-terminus of capsid protein 3. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: This product was cloned into the pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with HindIII and BamHI-HF (NEB), and the ∼1-kb fragment was extracted from an agarose gel and ligated into the similarly digested pET-30a expression vector (Novagen). .. The newly assembled plasmid, pJAM8, was transformed into chemically competent T7 Express competent E. coli (NEB C2566), and transformants were selected on LB with kanamycin.

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: Paragraph title: Cloning and expression of Mb-Fur ... After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector.

    Article Title: A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein
    Article Snippet: Paragraph title: Construction of TDsmURFP bacterial expression vector ... TDsmURFP was digested with BamHI-HF and EcoRI-HF (NEB), gel purified using Zymoclean Gel DNA Recovery Kit (Zymo), and subcloned into a pBAD (Life Technologies) vector containing HO-1 digested with BamHI-HF and EcoRI-HF (NEB) with T4 DNA Ligase (Life Technologies).

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
    Article Snippet: All coding regions were codon-optimized for E. coli expression. .. DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site. .. The resulting DNA constructs (verified by sequencing) were transformed into E. coli strain BL21 C41(DE3).

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: Antibody production and purification The scFvs of interest were converted into whole IgG4 antibodies by cloning the corresponding VH and VL cDNAs in the pEU vectors 8.2VH and 4.2VL, expressing respectively, the constant antibody heavy and light chains . .. In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector.

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: A recombinant plasmid expressing C. matruchotii MdbA under the control of the C. diphtheriae mdbA promoter was constructed as follows. .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ).

    Knock-In:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: The Gal4 knock-in cassette is described in detail in the . .. 10 ug of the resulting P[acman]-KO-1kb was digested with BamH1-HF (NEB R3136S) at 37°C for 6 hours.

    Transformation Assay:

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: This product was cloned into the pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with HindIII and BamHI-HF (NEB), and the ∼1-kb fragment was extracted from an agarose gel and ligated into the similarly digested pET-30a expression vector (Novagen).

    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
    Article Snippet: The amplification product was ligated into a pCR 2.1-TOPO plasmid vector and transformed into One Shot Top10 chemically competent E. coli , using a Topo TA kit (Life Technologies, Grand Island, NY). .. Plasmids were extracted using a PureLink Quick plasmid miniprep kit (Life Technologies) and then linearized with BamHI-HF enzyme (New England BioLabs, Ipswich, MA).

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. Expression of recombinant Mb-Fur was carried out in BL21 E. coli cells (Stratagene).

    Derivative Assay:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: The pIFF6 vector (gift by Franco Felici, Universita degli Studi del Molise, Italy) is derived from the pC89 vector and contains the coding sequence for the capsid protein pIII in place of pVIII [ ]. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Transfection:

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: Paragraph title: Transfection ... The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs).

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector. .. Stellar Competent Cells (Clontech Laboratories, 636,763) were transformed with the obtained vectors, and the colonies were screened by digestion and sequence analysis.

    Gas Chromatography:

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs). .. The green/blue vector was digested with XhoI and KpnI-HF (New England Biolabs) and the PCR amplified, XhoI and KpnI-HF digested Mxi-2 product (NM_139013, position 482 to 1431) was cloned (pEGFP/Mxi-2/EBFP).

    Serial Dilution:

    Article Title: A new genome-mining tool redefines the lasso peptide biosynthetic landscape
    Article Snippet: The DNA solution was split into 14 fractions and Sau3AI (NEB) was added to each in a 2-fold serial dilution, starting with 2.5 µL of Sau3AI into 400 µL of DNA solution. .. These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase.

    Infection:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Generated:

    Article Title: Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1
    Article Snippet: Cdc42, Rac1, Rac2, RhoA, k-Ras, and Rap1b were amplified from cDNA obtained from Missouri S & T cDNA Resource Center (Rolla, MO) and human Rasip1 and PKCɛ were amplified from cDNA obtained from Open Biosystems (Open Biosystems, GE Dharmacon, Lafayette, CO), and standard restriction digest cloning for individual GTPases and PKCɛ into pmCherry-C1 plasmid and Rasip1 into pAcGFP-C1 (Clontech, Mountain View, CA) using EcoRI-HF and BamHI-HF , XhoI and BamHI-HF , and EcoRI-HF and XbaI restriction enzymes respectively (New England Biolabs). .. Amplified S-Cherry, S-Ch-GTPase, S-Ch-PKCɛ, and AcGFP-Rasip1 constructs were subcloned into pShuttle-CMV expression plasmid using NotI-HF , XbaI , XhoI , KpnI-HF and SalI-HF restriction enzymes, respectively (New England Biolabs).

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: The Ctr9 knockout allele (Ctr9KO ) was generated by the method described previously ( ). .. This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008).

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: attB-P[acman]-KO was generated by inserting Frt/ISce1 site into the existing Pac1 and Asc1 sites of attB-P[acman] [ ] such that the original Pac1 and Asc1 sites were destroyed and new Asc1 and Pac1 sites generated proximally ( ). .. 10 ug of the resulting P[acman]-KO-1kb was digested with BamH1-HF (NEB R3136S) at 37°C for 6 hours.

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs). .. The green/blue vector was digested with XhoI and KpnI-HF (New England Biolabs) and the PCR amplified, XhoI and KpnI-HF digested Mxi-2 product (NM_139013, position 482 to 1431) was cloned (pEGFP/Mxi-2/EBFP).

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: Overlapping PCR was employed to link the promoter region to the coding sequence using both PCR products as the templates and primers pCmMdbA-HindIII-A and pCmMdbA-BamHI-D according to a published protocol ( ). .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ). .. The resulting plasmid was introduced into E. coli DH5α, and plasmid DNA was isolated for sequencing to confirm the cloned sequence.

    DNA Sequencing:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Article Title: Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation
    Article Snippet: Site-directed mutagenesis of Kv11.1 cDNA was performed using the QuikChange method (Agilent Technologies, Mulgrave, Victoria, Australia), and mutations were confirmed by Sanger DNA sequencing. .. WT and mutant channel cDNAs were linearized with BamHI-HF (New England Biolabs, Ipswich, MA), and cRNA was transcribed with T7 RNA polymerase using the mMessage mMachine kit (Ambion, Austin, TX).

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. The resultant Mb-Fur pHIS8 plasmid encodes for amino acids 1 to 411 of M. algicola Fur with an amino-terminal histidine tag and a thrombin cleavage site.

    Polymerase Chain Reaction:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Article Title: Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1
    Article Snippet: Cdc42, Rac1, Rac2, RhoA, k-Ras, and Rap1b were amplified from cDNA obtained from Missouri S & T cDNA Resource Center (Rolla, MO) and human Rasip1 and PKCɛ were amplified from cDNA obtained from Open Biosystems (Open Biosystems, GE Dharmacon, Lafayette, CO), and standard restriction digest cloning for individual GTPases and PKCɛ into pmCherry-C1 plasmid and Rasip1 into pAcGFP-C1 (Clontech, Mountain View, CA) using EcoRI-HF and BamHI-HF , XhoI and BamHI-HF , and EcoRI-HF and XbaI restriction enzymes respectively (New England Biolabs). .. Cdc42, Rac1, Rac2, RhoA, k-Ras, and Rap1b were amplified from cDNA obtained from Missouri S & T cDNA Resource Center (Rolla, MO) and human Rasip1 and PKCɛ were amplified from cDNA obtained from Open Biosystems (Open Biosystems, GE Dharmacon, Lafayette, CO), and standard restriction digest cloning for individual GTPases and PKCɛ into pmCherry-C1 plasmid and Rasip1 into pAcGFP-C1 (Clontech, Mountain View, CA) using EcoRI-HF and BamHI-HF , XhoI and BamHI-HF , and EcoRI-HF and XbaI restriction enzymes respectively (New England Biolabs).

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: The resulting 1 kb PCR product was cloned into the P[acman] KO vector using a Gateway BP reaction following the manufacturer’s instructions (Invitrogen BP clonase II 11789-020). .. This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008).

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: The 1kb PCR products were cloned into P[acman]-KO using the Gateway BP reaction following manufacturer's instruction (Invitrogen BP clonase II 11789-020). .. 10 ug of the resulting P[acman]-KO-1kb was digested with BamH1-HF (NEB R3136S) at 37°C for 6 hours.

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: This product was cloned into the pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with HindIII and BamHI-HF (NEB), and the ∼1-kb fragment was extracted from an agarose gel and ligated into the similarly digested pET-30a expression vector (Novagen).

    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
    Article Snippet: The amplification product was ligated into a pCR 2.1-TOPO plasmid vector and transformed into One Shot Top10 chemically competent E. coli , using a Topo TA kit (Life Technologies, Grand Island, NY). .. Plasmids were extracted using a PureLink Quick plasmid miniprep kit (Life Technologies) and then linearized with BamHI-HF enzyme (New England BioLabs, Ipswich, MA).

    Article Title: Bio-Inspired Liposomal Thrombomodulin Conjugate through Bio-Orthogonal Chemistry
    Article Snippet: Human thrombomodulin cDNA clone was from ATCC (Manassas, VA), pET Dsb Fusion System 39b, competent cells, and kanamycin sulfate were from EMD Chemicals (Philadelphia, PA). .. Phusion® High-Fidelity PCR Kit, BamHI-HF, KpnI-HF, T4 DNA ligase and alkaline phosphatase were from New England Biolabs (Ipswich, MA). .. All plasmid purification kits were from QIAGEN Inc. (Chatsworth, CA).

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: The complete fur gene ( 411 base pairs) was then amplified by standard PCR methods using Pfu polymerase and the primers 5′-TACTTATG CC GCGGCCGC TCAGGATTTGAGGGGTTTGA-3′and 5′-CACCTTCTAA GGATCC ATGTCATCCGA AAACACCGA-3′(restriction sites underlined). .. After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. The resultant Mb-Fur pHIS8 plasmid encodes for amino acids 1 to 411 of M. algicola Fur with an amino-terminal histidine tag and a thrombin cleavage site.

    Article Title: Radial glia regulate vascular patterning around the developing spinal cord
    Article Snippet: The gRNA was prepared in vitro using the MEGAshortscript T7 transcription kit (Ambion) after linearizing the plasmid with BamHI-HF (NEB). .. 1 nL of a solution containing 250 ng/µl of Cas9 mRNA and 100 ng/µl of gRNA was injected at the one-cell stage.

    Article Title: A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein
    Article Snippet: The two products were combined and TDsmURFP was created by bridging PCR with Phusion High-Fidelity DNA Polymerase (NEB). .. TDsmURFP was digested with BamHI-HF and EcoRI-HF (NEB), gel purified using Zymoclean Gel DNA Recovery Kit (Zymo), and subcloned into a pBAD (Life Technologies) vector containing HO-1 digested with BamHI-HF and EcoRI-HF (NEB) with T4 DNA Ligase (Life Technologies).

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs). .. The final product was a green/blue vector with a multiple cloning site (MCS) between both signals.

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: Briefly, the VHs and VLs were amplified by CloneAmp HiFi PCR Premix in standard conditions with specific primers and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, A9281) from 1,3% agarose gel. .. In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector.

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: Overlapping PCR was employed to link the promoter region to the coding sequence using both PCR products as the templates and primers pCmMdbA-HindIII-A and pCmMdbA-BamHI-D according to a published protocol ( ). .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ). .. The resulting plasmid was introduced into E. coli DH5α, and plasmid DNA was isolated for sequencing to confirm the cloned sequence.

    Sonication:

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
    Article Snippet: DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site. .. Cells were grown at 37 °C in 2× YT medium, which was supplemented with either 50 μg/ml CB or 100 μg/ml ampicillin, protein expression was induced with 0.1–0.3 mm IPTG at an A 600 nm of 0.6–0.7, and cells were cultured overnight at 22 °C.

    Injection:

    Article Title: Radial glia regulate vascular patterning around the developing spinal cord
    Article Snippet: The gRNA was prepared in vitro using the MEGAshortscript T7 transcription kit (Ambion) after linearizing the plasmid with BamHI-HF (NEB). .. The gRNA was purified using RNA Clean & Concentrator-5 kit.

    Recombinant:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: Abdurin libraries were subcloned in frame with pIII into pIFF6, a phagemid expression vector containing the pIII gene sequence of filamentous phage f1 for the expression of recombinant protein inserts at the amino-terminus of capsid protein 3. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Article Title: Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1
    Article Snippet: Cdc42, Rac1, Rac2, RhoA, k-Ras, and Rap1b were amplified from cDNA obtained from Missouri S & T cDNA Resource Center (Rolla, MO) and human Rasip1 and PKCɛ were amplified from cDNA obtained from Open Biosystems (Open Biosystems, GE Dharmacon, Lafayette, CO), and standard restriction digest cloning for individual GTPases and PKCɛ into pmCherry-C1 plasmid and Rasip1 into pAcGFP-C1 (Clontech, Mountain View, CA) using EcoRI-HF and BamHI-HF , XhoI and BamHI-HF , and EcoRI-HF and XbaI restriction enzymes respectively (New England Biolabs). .. Amplified S-Cherry, S-Ch-GTPase, S-Ch-PKCɛ, and AcGFP-Rasip1 constructs were subcloned into pShuttle-CMV expression plasmid using NotI-HF , XbaI , XhoI , KpnI-HF and SalI-HF restriction enzymes, respectively (New England Biolabs).

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008). .. This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008).

    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
    Article Snippet: Recombinant bacteria were enumerated on ImMedia ampicillin and kanamycin agar (Life Technologies), and colonies were selected randomly for overnight culture propagation in ImMedia broths (Life Technologies). .. Plasmids were extracted using a PureLink Quick plasmid miniprep kit (Life Technologies) and then linearized with BamHI-HF enzyme (New England BioLabs, Ipswich, MA).

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. The plasmid was isolated using a QIAGEN Plasmid Midi Kit and the correct gene sequence verified by DNA sequencing (Retogene).

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: A recombinant plasmid expressing C. matruchotii MdbA under the control of the C. diphtheriae mdbA promoter was constructed as follows. .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ).

    Molecular Weight:

    Article Title: A new genome-mining tool redefines the lasso peptide biosynthetic landscape
    Article Snippet: High molecular weight DNA was resuspended in 600 µL TE buffer (10 mM Tris, 1 mM EDTA, pH 8), 330 µL 10x CutSmart buffer (NEB), and 2310 µL DNase-free water. .. These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase.

    DNA Extraction:

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: After 1 and 3 h the samples were analyzed and DNA was isolated as described in DNA extraction. .. The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs).

    Nucleic Acid Electrophoresis:

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: The resulting 1 kb PCR product was cloned into the P[acman] KO vector using a Gateway BP reaction following the manufacturer’s instructions (Invitrogen BP clonase II 11789-020). .. This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008). .. This DNA was then electroporated into recombineering-competent DY380 cells carrying the 38M10 clone.

    Article Title: A new genome-mining tool redefines the lasso peptide biosynthetic landscape
    Article Snippet: Digest reactions were incubated at 37 °C for 1 h and were then inactivated by incubation at 75 °C for 30 min. Fractions were analyzed by field-inversion gel electrophoresis, and fractions containing approximately 40 kb pieces of DNA were utilized in cosmid construction. .. These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase.

    Article Title: Evolved Populations of Shigella flexneri Phage Sf6 Acquire Large Deletions, Altered Genomic Architecture, and Faster Life Cycles
    Article Snippet: Primers were constructed with Primer3Plus within Sf6 genes 1 (5′-AAGACTTCGCGTTGATACCC-3′, 5′-GTGCTCATCGGTGATGTTTC-3′), 5 (5′-TGCAACAGCAGACCAAACAG-3′, 5′-AAAGCGTAACCGTCACATCG-3′), 44 (5′-GACGCTGATGACGGCTATCA-3′, 5′-GCGTGTCGTCGATGTAAAGC-3′), and 61 (5′-GTTTGCAATGGCGTACCTTC-3′, 5′-CACATCGTTGCGTCGATTAC-3′) and were used with an in-house SYBR Green master mix as described ( ). .. Single restriction enzyme digests used BamHI HF and PstI HF (New England Biolabs) at 37°C for 15 min. Gel electrophoresis used 0.7% agarose in TAE and was run for 90 min at a constant 80 V. The ladder used was 1 kb Plus (Invitrogen). .. We conducted a phage evolution experiment for 10 replicate populations in which the phage, but not the host, were serially passaged on ompA − C − null Shigella , a host to which Sf6 only slowly adsorbs ( ) ( ).

    Gene Knockout:

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: The resulting 1 kb PCR product was cloned into the P[acman] KO vector using a Gateway BP reaction following the manufacturer’s instructions (Invitrogen BP clonase II 11789-020). .. This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008).

    Mutagenesis:

    Article Title: Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation
    Article Snippet: Site-directed mutagenesis of Kv11.1 cDNA was performed using the QuikChange method (Agilent Technologies, Mulgrave, Victoria, Australia), and mutations were confirmed by Sanger DNA sequencing. .. WT and mutant channel cDNAs were linearized with BamHI-HF (New England Biolabs, Ipswich, MA), and cRNA was transcribed with T7 RNA polymerase using the mMessage mMachine kit (Ambion, Austin, TX). .. Female X. laevis frogs were purchased from Nasco (Fort Atkinson, WI).

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ). .. The resulting plasmid was introduced into E. coli DH5α, and plasmid DNA was isolated for sequencing to confirm the cloned sequence.

    Isolation:

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: Genomic DNA was isolated from Marinobacter algicola via extraction with xanthate buffer ( ). .. After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector.

    Article Title: A new genome-mining tool redefines the lasso peptide biosynthetic landscape
    Article Snippet: Digest reactions were incubated at 37 °C for 1 h and were then inactivated by incubation at 75 °C for 30 min. Fractions were analyzed by field-inversion gel electrophoresis, and fractions containing approximately 40 kb pieces of DNA were utilized in cosmid construction. .. These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase. .. Cut vector was purified with a GeneJET PCR Purification Kit (Thermo Scientific).

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: After 1 and 3 h the samples were analyzed and DNA was isolated as described in DNA extraction. .. The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs).

    Purification:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: The pIFF6 vector (gift by Franco Felici, Universita degli Studi del Molise, Italy) is derived from the pC89 vector and contains the coding sequence for the capsid protein pIII in place of pVIII [ ]. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C. .. Ligation products were phenol extracted, ethanol precipitated and transformed into TOP10F’ electro-competent cells.

    Article Title: Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation
    Article Snippet: pT7-gRNA was linearized using BamHI-HF (NEB), containing the respective cloned target sites for either slc45a2 or tyr , were prepared using a QIAprep column (Qiagen) and transcribed using the MEGAscript T7 kit (Ambion) according to the manufacturer’s protocol. .. Prior to in-vitro transcription, the plasmid was linearized using XbaI (NEB) and cleaned up via a QIAprep Spin column.

    Article Title: Radial glia regulate vascular patterning around the developing spinal cord
    Article Snippet: The mRNA was purified using RNA Clean & Concentrator-5 kit (Zymo Research). .. The gRNA was prepared in vitro using the MEGAshortscript T7 transcription kit (Ambion) after linearizing the plasmid with BamHI-HF (NEB).

    Article Title: A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein
    Article Snippet: The two products were combined and TDsmURFP was created by bridging PCR with Phusion High-Fidelity DNA Polymerase (NEB). .. TDsmURFP was digested with BamHI-HF and EcoRI-HF (NEB), gel purified using Zymoclean Gel DNA Recovery Kit (Zymo), and subcloned into a pBAD (Life Technologies) vector containing HO-1 digested with BamHI-HF and EcoRI-HF (NEB) with T4 DNA Ligase (Life Technologies). .. Native PAGE was run using NativePAGE Novex Bis-Tris Gel System (Life Technologies) on NativePAGE 4–16% Bis-Tris protein gels (Life Technologies).

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
    Article Snippet: Paragraph title: Gene synthesis, cloning, protein expression, and purification for structural analysis ... DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site.

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: Paragraph title: Antibody production and purification ... In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector.

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: Overlapping PCR was employed to link the promoter region to the coding sequence using both PCR products as the templates and primers pCmMdbA-HindIII-A and pCmMdbA-BamHI-D according to a published protocol ( ). .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ). .. The resulting plasmid was introduced into E. coli DH5α, and plasmid DNA was isolated for sequencing to confirm the cloned sequence.

    Sequencing:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: The pIFF6 vector (gift by Franco Felici, Universita degli Studi del Molise, Italy) is derived from the pC89 vector and contains the coding sequence for the capsid protein pIII in place of pVIII [ ]. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C.

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. The resultant Mb-Fur pHIS8 plasmid encodes for amino acids 1 to 411 of M. algicola Fur with an amino-terminal histidine tag and a thrombin cleavage site.

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: Overlapping PCR was employed to link the promoter region to the coding sequence using both PCR products as the templates and primers pCmMdbA-HindIII-A and pCmMdbA-BamHI-D according to a published protocol ( ). .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ).

    CRISPR:

    Article Title: Radial glia regulate vascular patterning around the developing spinal cord
    Article Snippet: The gRNA was designed using Optimized CRISPR Design algorithm ( http://crispr.mit.edu ). .. The gRNA was prepared in vitro using the MEGAshortscript T7 transcription kit (Ambion) after linearizing the plasmid with BamHI-HF (NEB).

    IA:

    Article Title: Bio-Inspired Liposomal Thrombomodulin Conjugate through Bio-Orthogonal Chemistry
    Article Snippet: Phusion® High-Fidelity PCR Kit, BamHI-HF, KpnI-HF, T4 DNA ligase and alkaline phosphatase were from New England Biolabs (Ipswich, MA). .. Phusion® High-Fidelity PCR Kit, BamHI-HF, KpnI-HF, T4 DNA ligase and alkaline phosphatase were from New England Biolabs (Ipswich, MA).

    SDS Page:

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector. .. The conditioned media were collected, and the antibodies were purified by using Protein A HP SpinTrap (GE Healthcare Life Sciences, 28–9031–32).

    Plasmid Preparation:

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
    Article Snippet: The pIFF6 vector (gift by Franco Felici, Universita degli Studi del Molise, Italy) is derived from the pC89 vector and contains the coding sequence for the capsid protein pIII in place of pVIII [ ]. .. Briefly, 10 μg of DNA fragments for library #13, #15, #16 and #17 were digested with HF EcoRI /BamHI restriction enzymes (New England Biolabs, Boston, Massachusetts, USA), purified on Qiaquick columns (Qiagen, Hilden, Germany) and ligated into the EcoRI /BamHI dephosphorylated phagemid vector overnight at 16°C. .. Ligation products were phenol extracted, ethanol precipitated and transformed into TOP10F’ electro-competent cells.

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: The resulting 1 kb PCR product was cloned into the P[acman] KO vector using a Gateway BP reaction following the manufacturer’s instructions (Invitrogen BP clonase II 11789-020). .. This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008). .. This DNA was then electroporated into recombineering-competent DY380 cells carrying the 38M10 clone.

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: This product was cloned into the pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with HindIII and BamHI-HF (NEB), and the ∼1-kb fragment was extracted from an agarose gel and ligated into the similarly digested pET-30a expression vector (Novagen). .. The newly assembled plasmid, pJAM8, was transformed into chemically competent T7 Express competent E. coli (NEB C2566), and transformants were selected on LB with kanamycin.

    Article Title: Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats
    Article Snippet: Recombinant bacteria were enumerated on ImMedia ampicillin and kanamycin agar (Life Technologies), and colonies were selected randomly for overnight culture propagation in ImMedia broths (Life Technologies). .. Plasmids were extracted using a PureLink Quick plasmid miniprep kit (Life Technologies) and then linearized with BamHI-HF enzyme (New England BioLabs, Ipswich, MA). .. Linearized plasmid DNA was purified using a QIAquick PCR purification kit (Qiagen Inc., Valencia, CA) and quantified with a NanoDrop ND-1000 UV/Vis spectrophotometer (NanoDrop Technologies).

    Article Title: Bio-Inspired Liposomal Thrombomodulin Conjugate through Bio-Orthogonal Chemistry
    Article Snippet: Phusion® High-Fidelity PCR Kit, BamHI-HF, KpnI-HF, T4 DNA ligase and alkaline phosphatase were from New England Biolabs (Ipswich, MA). .. Phusion® High-Fidelity PCR Kit, BamHI-HF, KpnI-HF, T4 DNA ligase and alkaline phosphatase were from New England Biolabs (Ipswich, MA).

    Article Title: Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation
    Article Snippet: The cDNA of Kv11.1 (a gift from Dr. Gail Robertson, University of Wisconsin) was subcloned into a pBluescript vector, which contains the 5′-untranslated region (UTR) and 3′ UTR of the Xenopus laevis β-globin gene (a gift from Dr. Robert Vandenberg, University of Sydney). .. WT and mutant channel cDNAs were linearized with BamHI-HF (New England Biolabs, Ipswich, MA), and cRNA was transcribed with T7 RNA polymerase using the mMessage mMachine kit (Ambion, Austin, TX).

    Article Title: Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893
    Article Snippet: The complete fur gene ( 411 base pairs) was then amplified by standard PCR methods using Pfu polymerase and the primers 5′-TACTTATG CC GCGGCCGC TCAGGATTTGAGGGGTTTGA-3′and 5′-CACCTTCTAA GGATCC ATGTCATCCGA AAACACCGA-3′(restriction sites underlined). .. After cleanup using QIAquick Gel Extraction Kit (Qiagen), PCR amplicons were digested with BamHI-HF and NotI-HF (New England BioLabs) and ligated into a similarly double digested pHIS8 vector. .. The resultant Mb-Fur pHIS8 plasmid encodes for amino acids 1 to 411 of M. algicola Fur with an amino-terminal histidine tag and a thrombin cleavage site.

    Article Title: Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation
    Article Snippet: pT7-gRNA was linearized using BamHI-HF (NEB), containing the respective cloned target sites for either slc45a2 or tyr , were prepared using a QIAprep column (Qiagen) and transcribed using the MEGAscript T7 kit (Ambion) according to the manufacturer’s protocol. .. pT7-gRNA was linearized using BamHI-HF (NEB), containing the respective cloned target sites for either slc45a2 or tyr , were prepared using a QIAprep column (Qiagen) and transcribed using the MEGAscript T7 kit (Ambion) according to the manufacturer’s protocol.

    Article Title: Radial glia regulate vascular patterning around the developing spinal cord
    Article Snippet: The oligos (taggGTGTCAGCCGGCTGCAATAC and aaacGTATTGCAGCCGGCTGACAC) were annealed and ligated into the gRNA plasmid (pT7-gRNA vector) after digesting the plasmid with BsmbI (NEB). .. The gRNA was prepared in vitro using the MEGAshortscript T7 transcription kit (Ambion) after linearizing the plasmid with BamHI-HF (NEB). .. The gRNA was purified using RNA Clean & Concentrator-5 kit.

    Article Title: A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein
    Article Snippet: The two products were combined and TDsmURFP was created by bridging PCR with Phusion High-Fidelity DNA Polymerase (NEB). .. TDsmURFP was digested with BamHI-HF and EcoRI-HF (NEB), gel purified using Zymoclean Gel DNA Recovery Kit (Zymo), and subcloned into a pBAD (Life Technologies) vector containing HO-1 digested with BamHI-HF and EcoRI-HF (NEB) with T4 DNA Ligase (Life Technologies). .. Native PAGE was run using NativePAGE Novex Bis-Tris Gel System (Life Technologies) on NativePAGE 4–16% Bis-Tris protein gels (Life Technologies).

    Article Title: A new genome-mining tool redefines the lasso peptide biosynthetic landscape
    Article Snippet: Digest reactions were incubated at 37 °C for 1 h and were then inactivated by incubation at 75 °C for 30 min. Fractions were analyzed by field-inversion gel electrophoresis, and fractions containing approximately 40 kb pieces of DNA were utilized in cosmid construction. .. These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase. .. Cut vector was purified with a GeneJET PCR Purification Kit (Thermo Scientific).

    Article Title: Beyond the 3′UTR binding–microRNA-induced protein truncation via DNA binding
    Article Snippet: A double-labelled vector was constructed for pEGFP/EBFP analysis. .. The commercially available vectors pEGFP and pEBFP (Clontech, Saint-Germain-en-Laye, France) were digested with AseI and BamHI-HF (New England Biolabs).

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3
    Article Snippet: All coding regions were codon-optimized for E. coli expression. .. DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site. .. The resulting DNA constructs (verified by sequencing) were transformed into E. coli strain BL21 C41(DE3).

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: Briefly, the VHs and VLs were amplified by CloneAmp HiFi PCR Premix in standard conditions with specific primers and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, A9281) from 1,3% agarose gel. .. In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector. .. Stellar Competent Cells (Clontech Laboratories, 636,763) were transformed with the obtained vectors, and the colonies were screened by digestion and sequence analysis.

    Article Title: Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii
    Article Snippet: A recombinant plasmid expressing C. matruchotii MdbA under the control of the C. diphtheriae mdbA promoter was constructed as follows. .. The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 ( ).

    Positron Emission Tomography:

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: This product was cloned into the pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with HindIII and BamHI-HF (NEB), and the ∼1-kb fragment was extracted from an agarose gel and ligated into the similarly digested pET-30a expression vector (Novagen). .. The newly assembled plasmid, pJAM8, was transformed into chemically competent T7 Express competent E. coli (NEB C2566), and transformants were selected on LB with kanamycin.

    Article Title: Bio-Inspired Liposomal Thrombomodulin Conjugate through Bio-Orthogonal Chemistry
    Article Snippet: Human thrombomodulin cDNA clone was from ATCC (Manassas, VA), pET Dsb Fusion System 39b, competent cells, and kanamycin sulfate were from EMD Chemicals (Philadelphia, PA). .. Phusion® High-Fidelity PCR Kit, BamHI-HF, KpnI-HF, T4 DNA ligase and alkaline phosphatase were from New England Biolabs (Ipswich, MA).

    Agarose Gel Electrophoresis:

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: This product was cloned into the pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with HindIII and BamHI-HF (NEB), and the ∼1-kb fragment was extracted from an agarose gel and ligated into the similarly digested pET-30a expression vector (Novagen). .. The newly assembled plasmid, pJAM8, was transformed into chemically competent T7 Express competent E. coli (NEB C2566), and transformants were selected on LB with kanamycin.

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: Briefly, the VHs and VLs were amplified by CloneAmp HiFi PCR Premix in standard conditions with specific primers and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, A9281) from 1,3% agarose gel. .. In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector.

    In Vitro:

    Article Title: Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation
    Article Snippet: Paragraph title: In-vitro transcription of gRNA and Cas9 mRNA ... pT7-gRNA was linearized using BamHI-HF (NEB), containing the respective cloned target sites for either slc45a2 or tyr , were prepared using a QIAprep column (Qiagen) and transcribed using the MEGAscript T7 kit (Ambion) according to the manufacturer’s protocol.

    Article Title: Radial glia regulate vascular patterning around the developing spinal cord
    Article Snippet: The oligos (taggGTGTCAGCCGGCTGCAATAC and aaacGTATTGCAGCCGGCTGACAC) were annealed and ligated into the gRNA plasmid (pT7-gRNA vector) after digesting the plasmid with BsmbI (NEB). .. The gRNA was prepared in vitro using the MEGAshortscript T7 transcription kit (Ambion) after linearizing the plasmid with BamHI-HF (NEB). .. The gRNA was purified using RNA Clean & Concentrator-5 kit.

    Knock-Out:

    Article Title: Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development
    Article Snippet: Paragraph title: Generation of the Ctr9 knockout ... This vector was then linearized using BAMHI-HF (NEB R3136S) at 37° for 6 hr, subjected to gel electrophoresis, and gel extracted using a Zymoclean Gel DNA recovery kit (Zymo Research D4008).

    Produced:

    Article Title: Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation
    Article Snippet: pT7-gRNA was linearized using BamHI-HF (NEB), containing the respective cloned target sites for either slc45a2 or tyr , were prepared using a QIAprep column (Qiagen) and transcribed using the MEGAscript T7 kit (Ambion) according to the manufacturer’s protocol. .. Prior to in-vitro transcription, the plasmid was linearized using XbaI (NEB) and cleaned up via a QIAprep Spin column.

    Concentration Assay:

    Article Title: A new genome-mining tool redefines the lasso peptide biosynthetic landscape
    Article Snippet: These fractions were dephosphorylated by the addition of 20 µL of shrimp alkaline phosphatase and incubation at 37 °C for 1 h. DNA fragments were isolated by phenol:chloroform:isoamyl alcohol extraction and air dried for 3 h. The previously reported vector pJK050 was used for fosmid construction . pJK050 was doubly digested with NheI-HF and BamHI-HF (NEB) and dephosphorylated with shrimp alkaline phosphatase. .. Approximately 1 µg of cut vector, 2 µL of 10x T4 ligase buffer (NEB), and water (to give 20 µL total volume) was used to resuspend the digested high molecular weight DNA.

    Staining:

    Article Title: Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies
    Article Snippet: In-Fusion HD cloning kit (Clontech Laboratories, 639,692) was used to clone the VHs in Bam HI (R3136) and Bss HII (R0199) all from New England Biolabs, linearized pEU8.2VH vector, and the VLs in Apa LI (R0507) and Avr II (R0174) New England Biolabs, linearized pEU4.2VL vector. .. The conditioned media were collected, and the antibodies were purified by using Protein A HP SpinTrap (GE Healthcare Life Sciences, 28–9031–32).

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    New England Biolabs bamhi buffer
    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_MEM_3rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_MEM_3rRFBs+ (8908 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note the insertion of an EcoRI fragment containing the three putative Fob1 binding sites expected to act as RFBs (indicated in red), described by Kobayashi ( 20 ). ( B ) Map of the 3708 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with SwaI and <t>BamHI</t> showing the relative position of the three putative RFBs. ( C ) Map of the 5186 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with <t>EcoRV</t> and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3708 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5186-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them.
    Bamhi Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_MEM_3rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_MEM_3rRFBs+ (8908 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note the insertion of an EcoRI fragment containing the three putative Fob1 binding sites expected to act as RFBs (indicated in red), described by Kobayashi ( 20 ). ( B ) Map of the 3708 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with SwaI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 5186 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3708 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5186-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them.

    Journal: Nucleic Acids Research

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

    doi: 10.1093/nar/gkx655

    Figure Lengend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_MEM_3rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_MEM_3rRFBs+ (8908 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note the insertion of an EcoRI fragment containing the three putative Fob1 binding sites expected to act as RFBs (indicated in red), described by Kobayashi ( 20 ). ( B ) Map of the 3708 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with SwaI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 5186 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3708 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5186-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them.

    Article Snippet: DNA was digested with the restriction endonucleases BamHI, EcoRV, SwaI, FspI and MluI (New England Biolabs) for at least 2 h at 37°C except for SwaI that was digested at 25°C.

    Techniques: Two-Dimensional Gel Electrophoresis, Binding Assay, Activated Clotting Time Assay, Generated

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_3′rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_3′rRFBs+ (8175 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the three putative Fob1 binding sites expected to act as RFBs (indicated in red and white), described by Kobayashi ( 20 ), are equally distanced. ( B ) Map of the 3710-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with FspI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 4454-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the FspI-BamHI 3710-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4454-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them. For comparison, the densitometric profile corresponding to pYAC_MEM_3rRFBs shown in Figure 4H is presented on top.

    Journal: Nucleic Acids Research

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

    doi: 10.1093/nar/gkx655

    Figure Lengend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_3′rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_3′rRFBs+ (8175 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the three putative Fob1 binding sites expected to act as RFBs (indicated in red and white), described by Kobayashi ( 20 ), are equally distanced. ( B ) Map of the 3710-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with FspI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 4454-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the FspI-BamHI 3710-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4454-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them. For comparison, the densitometric profile corresponding to pYAC_MEM_3rRFBs shown in Figure 4H is presented on top.

    Article Snippet: DNA was digested with the restriction endonucleases BamHI, EcoRV, SwaI, FspI and MluI (New England Biolabs) for at least 2 h at 37°C except for SwaI that was digested at 25°C.

    Techniques: Two-Dimensional Gel Electrophoresis, Binding Assay, Activated Clotting Time Assay, Generated

    Genetic map and 2D gel analysis of linear fragments corresponding to pYAC_MEM. ( A ) Genetic map of pYAC_MEM (7966 bp) showing its most relevant features: clockwise starting with the replication origin ARS4 (indicated in green), URA3 gene active in Saccharomyces cerevisiae (indicated in light blue), L1 lambda DNA used for hybridization (indicated in yellow), HIS3 gene active in S. cerevisiae (indicated in light blue), L2 lambda DNA used for hybridization (indicated in yellow), the ColE1 replication origin active only in Escherichia coli (indicated in gray), the ampicillin resistance gene active only in E. coli (indicated in gray) and the budding yeast centromeric sequence CEN6 (indicated in orange). The sites for specific restriction endonucleases are indicated outside the map. In addition, a magenta triangle points the position located 180° apart from the replication origin ARS4. ( B ) Map of the 2764-bp linear fragment generated by digestion of pYAC_MEM with SwaI and BamHI. ( C ) Map of the 4245-bp linear fragment generated by digestion of pYAC_MEM with EcoRV and MluI. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 2764 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4245-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). De-localized termination signals are indicated in magenta.

    Journal: Nucleic Acids Research

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

    doi: 10.1093/nar/gkx655

    Figure Lengend Snippet: Genetic map and 2D gel analysis of linear fragments corresponding to pYAC_MEM. ( A ) Genetic map of pYAC_MEM (7966 bp) showing its most relevant features: clockwise starting with the replication origin ARS4 (indicated in green), URA3 gene active in Saccharomyces cerevisiae (indicated in light blue), L1 lambda DNA used for hybridization (indicated in yellow), HIS3 gene active in S. cerevisiae (indicated in light blue), L2 lambda DNA used for hybridization (indicated in yellow), the ColE1 replication origin active only in Escherichia coli (indicated in gray), the ampicillin resistance gene active only in E. coli (indicated in gray) and the budding yeast centromeric sequence CEN6 (indicated in orange). The sites for specific restriction endonucleases are indicated outside the map. In addition, a magenta triangle points the position located 180° apart from the replication origin ARS4. ( B ) Map of the 2764-bp linear fragment generated by digestion of pYAC_MEM with SwaI and BamHI. ( C ) Map of the 4245-bp linear fragment generated by digestion of pYAC_MEM with EcoRV and MluI. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 2764 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4245-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). De-localized termination signals are indicated in magenta.

    Article Snippet: DNA was digested with the restriction endonucleases BamHI, EcoRV, SwaI, FspI and MluI (New England Biolabs) for at least 2 h at 37°C except for SwaI that was digested at 25°C.

    Techniques: Two-Dimensional Gel Electrophoresis, Lambda DNA Preparation, Hybridization, Sequencing, Generated

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ isolated from cells that overexpress Fob1 and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the ten putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194 bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in ( D ) is presented in ( H ) indicating the height of the peaks. For comparison, the densitometric profile corresponding to the 3705-bp SwaI-BamHI of pYAC_AC_10rRFBs isolated from the top2-td strain shown in Figure 4H is presented on top.

    Journal: Nucleic Acids Research

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

    doi: 10.1093/nar/gkx655

    Figure Lengend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ isolated from cells that overexpress Fob1 and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the ten putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194 bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in ( D ) is presented in ( H ) indicating the height of the peaks. For comparison, the densitometric profile corresponding to the 3705-bp SwaI-BamHI of pYAC_AC_10rRFBs isolated from the top2-td strain shown in Figure 4H is presented on top.

    Article Snippet: DNA was digested with the restriction endonucleases BamHI, EcoRV, SwaI, FspI and MluI (New England Biolabs) for at least 2 h at 37°C except for SwaI that was digested at 25°C.

    Techniques: Two-Dimensional Gel Electrophoresis, Isolation, Binding Assay, Activated Clotting Time Assay, In Vivo, Generated

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the 10 putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks.

    Journal: Nucleic Acids Research

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

    doi: 10.1093/nar/gkx655

    Figure Lengend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the 10 putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks.

    Article Snippet: DNA was digested with the restriction endonucleases BamHI, EcoRV, SwaI, FspI and MluI (New England Biolabs) for at least 2 h at 37°C except for SwaI that was digested at 25°C.

    Techniques: Two-Dimensional Gel Electrophoresis, Binding Assay, Activated Clotting Time Assay, In Vivo, Generated

    Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Plasmid Preparation, Incubation

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    Digestion of T7 genomic DNA by variant M91V/E156K. ( A ) A virtual digest of T7 DNA using NEBcutter ( 29 ). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. ( B ) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. ( C ) A control showing no digestion of T7 DNA by 200 U wt NotI.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Digestion of T7 genomic DNA by variant M91V/E156K. ( A ) A virtual digest of T7 DNA using NEBcutter ( 29 ). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. ( B ) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. ( C ) A control showing no digestion of T7 DNA by 200 U wt NotI.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Marker

    Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Derivative Assay, Incubation

    Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Journal: PLoS ONE

    Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells

    doi: 10.1371/journal.pone.0141172

    Figure Lengend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Article Snippet: To perform sub-cloning, Hras V12 and SV40 large T antigene (LTg) were separated from pBABE-Hras V12 and pBABE-SV40 LTg by the enzymatic digestion with BamHI and EcoRI (NEB, Ipswich, MA), or with BamHI (NEB), respectively.

    Techniques: Subcloning, Clone Assay, DNA Sequencing

    pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.

    Journal: PLoS ONE

    Article Title: TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

    doi: 10.1371/journal.pone.0186568

    Figure Lengend Snippet: pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.

    Article Snippet: The purified pET-BccI batches were analyzed through the digestion of 1 μg plasmid DNA with 1 unit of the restriction endonucleases BccI, EcoRI, BamHI and HindIII (New England BioLabs), for 1 hour, at 37°C in the NEB buffers: CutSmart, EcoRI, 3.1 and 2.1, respectively.

    Techniques: Positron Emission Tomography

    Screening for E . coli colonies transformed with recombinant pET-BccI. Plasmid preparations from cultures inoculated with transformed colonies were digested with BamHI and subjected to electrophoresis. A ) For BRP recombinants pET-BccI screening, the presence of a 283 bp DNA band indicated there was no recombination (A3, A7, A9), whereas the presence of a 636 bp insert DNA band showed a successful recombination (A2, A4, A5, A6, A8). B ) For CAT recombinants pET-BccI screening, again the presence of a 283 bp DNA band indicated there was no recombination (B2, B5, B6, B8), whereas the presence of a 924 bp insert DNA band showed a successful recombination (B3, B4, B7, B9).

    Journal: PLoS ONE

    Article Title: TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

    doi: 10.1371/journal.pone.0186568

    Figure Lengend Snippet: Screening for E . coli colonies transformed with recombinant pET-BccI. Plasmid preparations from cultures inoculated with transformed colonies were digested with BamHI and subjected to electrophoresis. A ) For BRP recombinants pET-BccI screening, the presence of a 283 bp DNA band indicated there was no recombination (A3, A7, A9), whereas the presence of a 636 bp insert DNA band showed a successful recombination (A2, A4, A5, A6, A8). B ) For CAT recombinants pET-BccI screening, again the presence of a 283 bp DNA band indicated there was no recombination (B2, B5, B6, B8), whereas the presence of a 924 bp insert DNA band showed a successful recombination (B3, B4, B7, B9).

    Article Snippet: The purified pET-BccI batches were analyzed through the digestion of 1 μg plasmid DNA with 1 unit of the restriction endonucleases BccI, EcoRI, BamHI and HindIII (New England BioLabs), for 1 hour, at 37°C in the NEB buffers: CutSmart, EcoRI, 3.1 and 2.1, respectively.

    Techniques: Transformation Assay, Recombinant, Positron Emission Tomography, Plasmid Preparation, Electrophoresis

    The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.

    Journal: PLoS ONE

    Article Title: TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

    doi: 10.1371/journal.pone.0186568

    Figure Lengend Snippet: The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.

    Article Snippet: The purified pET-BccI batches were analyzed through the digestion of 1 μg plasmid DNA with 1 unit of the restriction endonucleases BccI, EcoRI, BamHI and HindIII (New England BioLabs), for 1 hour, at 37°C in the NEB buffers: CutSmart, EcoRI, 3.1 and 2.1, respectively.

    Techniques: Expressing, Plasmid Preparation, Positron Emission Tomography, Derivative Assay, Selection, Transformation Assay, Clone Assay, Recombinant, Ligation