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  • 98
    New England Biolabs vent dna polymerase
    Targeting strategy for inactivation of the mouse Daf genes. The tandem <t>Daf1</t> and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the <t>DNA</t> fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.
    Vent Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 20791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs vent exo dna polymerase
    Ligation of short padlock probes. Ligation products ( A ) and the <t>Exo</t> III and -VII digestion mixtures ( B ) were separated on 12% denaturing polyacrylamide gels. <t>DNA</t> was revealed using a standard silver-staining method. G and A represent the target genotypes GG and AA from S75 and mutant 109, respectively. M is the 30–330 bp AFLP DNA Ladder (Life Technologies). (A) Linear padlock probes and circularised padlock probes are indicated by solid arrows and asterisks, respectively. (B) After ligation, 5 µl of digestion solution containing 100 U of Exonuclease III and 0.75 U of Exonuclease VII was added to the ligation mixture. Digestion was carried out at 37°C for 45 min and the reaction stopped by heating to 75°C for 10 min. After digestion, only circularised padlock probes derived from the allele-specific ligations remained.
    Vent Exo Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs deep vent dna polymerase
    BB0347 is expressed and produced in culture. A) Expression of BB0347 was verified by RT- <t>PCR</t> with flaB mRNA as a control. Lane 1: flaB from genomic <t>DNA,</t> lane 2: flaB from cDNA, lane 3: no RT control, lane 4: bb0347 from genomic DNA, lane 5: bb0347 from cDNA, lane 6: no RT control, L: ladder. B) Western blotting shows that BB0347 protein is produced in the spirochete at all sampled time points. C) QRT- PCR of bb0347 at two different temperatures of incubation with flaB as a standard. D) Western blot using αBB0347 and αFlaB against whole-cell lysates from spirochetes grown at either 34 or 23°C to similar cellular densities. All figures are representative of at least two independent experiments with similar results, and error bars indicate ±SEM.
    Deep Vent Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 986 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dna polymerase
    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed <t>DNA</t> sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and <t>dNTP.</t> The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.
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    96
    New England Biolabs deep vent exo polymerase
    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed <t>DNA</t> sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and <t>dNTP.</t> The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.
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    New England Biolabs dna modifying enzymes
    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed <t>DNA</t> sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and <t>dNTP.</t> The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.
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    Image Search Results


    Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.

    Journal: Immunology

    Article Title: Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies

    doi: 10.1046/j.0019-2805.2001.01287.x

    Figure Lengend Snippet: Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.

    Article Snippet: The coding regions for mouse DAF CCP1-4, CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were amplified from mouse Daf1 cDNA with proofreading Vent DNA polymerase (New England Biolabs, Beverly, MA).

    Techniques: Selection, Marker, Construct, Introduce, Knock-Out

    PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/ loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5 , P6 ( Daf1 -specific) and P7 ( Daf2 -specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1 −/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, Bam HI; E, Eco RI; and S, Sac I. The position of the 1·5-kb Sac I probe is indicated and the hybridized Eco RI and Bam HI fragments are shown by the brackets. (c) Southern blot analyses of Eco RI- and Bam HI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous ( Daf1 +/− ) and homozygous ( Daf1 −/− ) knock-out mice were hybridized with the Sac I fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high M r band corresponds to the homologous Eco RI fragment in the Daf2 gene.

    Journal: Immunology

    Article Title: Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies

    doi: 10.1046/j.0019-2805.2001.01287.x

    Figure Lengend Snippet: PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/ loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5 , P6 ( Daf1 -specific) and P7 ( Daf2 -specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1 −/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, Bam HI; E, Eco RI; and S, Sac I. The position of the 1·5-kb Sac I probe is indicated and the hybridized Eco RI and Bam HI fragments are shown by the brackets. (c) Southern blot analyses of Eco RI- and Bam HI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous ( Daf1 +/− ) and homozygous ( Daf1 −/− ) knock-out mice were hybridized with the Sac I fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high M r band corresponds to the homologous Eco RI fragment in the Daf2 gene.

    Article Snippet: The coding regions for mouse DAF CCP1-4, CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were amplified from mouse Daf1 cDNA with proofreading Vent DNA polymerase (New England Biolabs, Beverly, MA).

    Techniques: Polymerase Chain Reaction, Construct, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Sequencing, Southern Blot, Knock-Out

    PCR amplification of DNA fragments containing two Ds bases (60-, 62-, 65- or 68-mer). ( a ) Scheme for the PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. The amplified DNA fragments containing FAM-hx- Px were detected by denaturing PAGE. ( b ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on the gel was detected with an FLA-7000 bio-imager, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. ( c ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of NH 2 -hx-d Px TP and d Ds TP. ( d ) Sequencing of the 15-cycle amplified DNA fragments, in the presence of d Pa ′TP (50 μM). The PCR products amplified in the presence of NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds -strands. The arrows indicate the unnatural base position.

    Journal: Nucleic Acids Research

    Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    doi: 10.1093/nar/gkn956

    Figure Lengend Snippet: PCR amplification of DNA fragments containing two Ds bases (60-, 62-, 65- or 68-mer). ( a ) Scheme for the PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. The amplified DNA fragments containing FAM-hx- Px were detected by denaturing PAGE. ( b ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on the gel was detected with an FLA-7000 bio-imager, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. ( c ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of NH 2 -hx-d Px TP and d Ds TP. ( d ) Sequencing of the 15-cycle amplified DNA fragments, in the presence of d Pa ′TP (50 μM). The PCR products amplified in the presence of NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds -strands. The arrows indicate the unnatural base position.

    Article Snippet: After five rounds of in vitro selection, about 1 pmol of the recovered DNA was amplified by eight cycles of PCR (25 μl), in a reaction buffer with 50 μM NH2 -hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP, 1 μM each of 5′-primer (40-mer, 5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3′) and 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.02 U/μl DeepVent DNA polymerase, and then the PCR products were purified by electrophoresis on 8% polyacrylamide—7 M urea gel for direct sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Labeling, Fluorescence, Radioactivity, Autoradiography, Sequencing

    PCR amplification of DNA fragments (55-mer) containing one Ds base. ( a ) Scheme for PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. To detect DNA fragments containing FAM-hx- Px , the PCR products were analyzed by denaturing PAGE. DNA fragments amplified with NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds strands. ( b ) Polyacrylamide-gel analysis of the DNA fragments amplified by 15 or 30 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on a gel was detected with a bio-imaging analyzer, FLA-7000, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. The fold amplification of each DNA fragment is summarized in Table S3. ( c ) Sequencing of the 40-cycle amplified DNA fragments, in the presence of d Pa′ TP (2 μM) or dd Pa′ TP (50 μM). The arrows indicate the unnatural base position.

    Journal: Nucleic Acids Research

    Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    doi: 10.1093/nar/gkn956

    Figure Lengend Snippet: PCR amplification of DNA fragments (55-mer) containing one Ds base. ( a ) Scheme for PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. To detect DNA fragments containing FAM-hx- Px , the PCR products were analyzed by denaturing PAGE. DNA fragments amplified with NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds strands. ( b ) Polyacrylamide-gel analysis of the DNA fragments amplified by 15 or 30 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on a gel was detected with a bio-imaging analyzer, FLA-7000, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. The fold amplification of each DNA fragment is summarized in Table S3. ( c ) Sequencing of the 40-cycle amplified DNA fragments, in the presence of d Pa′ TP (2 μM) or dd Pa′ TP (50 μM). The arrows indicate the unnatural base position.

    Article Snippet: After five rounds of in vitro selection, about 1 pmol of the recovered DNA was amplified by eight cycles of PCR (25 μl), in a reaction buffer with 50 μM NH2 -hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP, 1 μM each of 5′-primer (40-mer, 5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3′) and 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.02 U/μl DeepVent DNA polymerase, and then the PCR products were purified by electrophoresis on 8% polyacrylamide—7 M urea gel for direct sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Sequencing, Labeling, Fluorescence, Imaging, Radioactivity, Autoradiography

    Overview of in vitro selection using a Ds -containing DNA library. ( a ) Scheme for in vitro selection using the Ds -containing DNA library, by FAM-hx- Px incorporation into PCR products and isolation with an anti-fluorescein antibody. A chemically synthesized, single-stranded DNA library containing an NNN Ds NNN sequence (55-mer) was amplified by 10 cycles of PCR, in the presence of natural dNTPs, d Ds TP and FAM-hx-d Px TP, with DeepVent DNA Pol. After selection of the PCR products containing FAM-hx- Px by binding with an anti-fluorescein antibody, the isolated DNA fragments were used as a template for the next round of PCR amplification and selection. For direct sequencing of the library after five rounds of selection, the isolated DNA fragments were amplified in the presence of NH 2 -hx-d Px TP, instead of FAM-hx-d Px TP ( Figure 3 a), by eight cycles of PCR. Direct sequencing was performed in the presence of 2 μM d Pa′ TP, using a BigDye terminator v1.1 Cycle Sequencing kit. For the sequencing of clones after five rounds of selection, the library was amplified in the presence of only the natural dNTPs with Taq DNA polymerase, and the PCR products were used for TOPO TA cloning. ( b ) Sequencing of the DNA library after five rounds of selection. ( c ) Sequencing of the initial library. The arrow indicates the unnatural base position. ( d ) Probability (%) of occurrence at each position of the selected 66-clone sequences ( Supplementary Figure 1 ). Bases with an occurrence rate of ≥35% among the clones are colored red.

    Journal: Nucleic Acids Research

    Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    doi: 10.1093/nar/gkn956

    Figure Lengend Snippet: Overview of in vitro selection using a Ds -containing DNA library. ( a ) Scheme for in vitro selection using the Ds -containing DNA library, by FAM-hx- Px incorporation into PCR products and isolation with an anti-fluorescein antibody. A chemically synthesized, single-stranded DNA library containing an NNN Ds NNN sequence (55-mer) was amplified by 10 cycles of PCR, in the presence of natural dNTPs, d Ds TP and FAM-hx-d Px TP, with DeepVent DNA Pol. After selection of the PCR products containing FAM-hx- Px by binding with an anti-fluorescein antibody, the isolated DNA fragments were used as a template for the next round of PCR amplification and selection. For direct sequencing of the library after five rounds of selection, the isolated DNA fragments were amplified in the presence of NH 2 -hx-d Px TP, instead of FAM-hx-d Px TP ( Figure 3 a), by eight cycles of PCR. Direct sequencing was performed in the presence of 2 μM d Pa′ TP, using a BigDye terminator v1.1 Cycle Sequencing kit. For the sequencing of clones after five rounds of selection, the library was amplified in the presence of only the natural dNTPs with Taq DNA polymerase, and the PCR products were used for TOPO TA cloning. ( b ) Sequencing of the DNA library after five rounds of selection. ( c ) Sequencing of the initial library. The arrow indicates the unnatural base position. ( d ) Probability (%) of occurrence at each position of the selected 66-clone sequences ( Supplementary Figure 1 ). Bases with an occurrence rate of ≥35% among the clones are colored red.

    Article Snippet: After five rounds of in vitro selection, about 1 pmol of the recovered DNA was amplified by eight cycles of PCR (25 μl), in a reaction buffer with 50 μM NH2 -hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP, 1 μM each of 5′-primer (40-mer, 5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3′) and 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.02 U/μl DeepVent DNA polymerase, and then the PCR products were purified by electrophoresis on 8% polyacrylamide—7 M urea gel for direct sequencing.

    Techniques: In Vitro, Selection, Polymerase Chain Reaction, Isolation, Synthesized, Sequencing, Amplification, Binding Assay, Clone Assay, TA Cloning

    Agarose gel electrophoresis of PCR products from amplification of MPtV-related sequences in mouse DNA. DNA was extracted from the spleen of a C3H mouse and then purified. PCR amplification was done for 35 cycles in the presence or absence (−) of template DNA by using a primer pair that would generate a 179-bp product with the in A or in B insert of MPtV DNA as a template. After amplification, DNA was resolved by electrophoresis in a 1.5% agarose gel in the presence of 0.5 μg of ethidium bromide per ml. DNA was visualized in UV light. The final concentration of MgCl 2 in the PCR is indicated. The electrophoretic mobility of size markers is displayed to the left.

    Journal: Journal of Virology

    Article Title: Cellular Mobile Genetic Elements in the Regulatory Region of the Pneumotropic Mouse Polyomavirus Genome: Structure and Function in Viral Gene Expression and DNA Replication

    doi: 10.1128/JVI.77.6.3477-3486.2003

    Figure Lengend Snippet: Agarose gel electrophoresis of PCR products from amplification of MPtV-related sequences in mouse DNA. DNA was extracted from the spleen of a C3H mouse and then purified. PCR amplification was done for 35 cycles in the presence or absence (−) of template DNA by using a primer pair that would generate a 179-bp product with the in A or in B insert of MPtV DNA as a template. After amplification, DNA was resolved by electrophoresis in a 1.5% agarose gel in the presence of 0.5 μg of ethidium bromide per ml. DNA was visualized in UV light. The final concentration of MgCl 2 in the PCR is indicated. The electrophoretic mobility of size markers is displayed to the left.

    Article Snippet: The PCR was performed with the high-fidelity Vent DNA polymerase (New England Biolabs).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Purification, Electrophoresis, Concentration Assay

    Analysis of MPtV genome structure. (A) The regulatory region of MPtV DNA extracted from a crude virus preparation (VS) was amplified by PCR by using primers complementary to the region immediately adjacent to protein coding sequences. As a control DNA from the plasmid p st MPtV (VP) was used. The amplification products were separated by agarose gel electrophoresis. (B) Southern blot analysis of Xba I-digested DNA from lung extracts (VS) and from p st MPtV (VP). Annealing was done with 32 P-labeled MPtV DNA isolated from the recombinant plasmid p st MPtV, which was used as a probe. The mobility of DNA size markers is indicated to the left. kb, kilobase pairs.

    Journal: Journal of Virology

    Article Title: Cellular Mobile Genetic Elements in the Regulatory Region of the Pneumotropic Mouse Polyomavirus Genome: Structure and Function in Viral Gene Expression and DNA Replication

    doi: 10.1128/JVI.77.6.3477-3486.2003

    Figure Lengend Snippet: Analysis of MPtV genome structure. (A) The regulatory region of MPtV DNA extracted from a crude virus preparation (VS) was amplified by PCR by using primers complementary to the region immediately adjacent to protein coding sequences. As a control DNA from the plasmid p st MPtV (VP) was used. The amplification products were separated by agarose gel electrophoresis. (B) Southern blot analysis of Xba I-digested DNA from lung extracts (VS) and from p st MPtV (VP). Annealing was done with 32 P-labeled MPtV DNA isolated from the recombinant plasmid p st MPtV, which was used as a probe. The mobility of DNA size markers is indicated to the left. kb, kilobase pairs.

    Article Snippet: The PCR was performed with the high-fidelity Vent DNA polymerase (New England Biolabs).

    Techniques: Amplification, Polymerase Chain Reaction, Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Labeling, Isolation, Recombinant

    In vivo DMS footprinting of the lower strand of the TRE-1s. DNAs from in vivo DMS-treated MT-2 and MT-4 cells were piperidine cleaved and subjected to LMPCR as described in Materials and Methods. The reaction products were separated on polyacrylamide gels and exposed to film. Autoradiograms corresponding to the three TRE-1s are shown. (A) Promoter-distal TRE-1. A band is missing in the 5′ GC-rich flank most likely because the proviral DNA contains a base other than the predicted guanine residue at that position. (B) Promoter-central TRE-1. (C) Promoter-proximal TRE-1. Arrowheads pointing toward bands indicate hypersensitive residues; arrowheads pointing away from bands indicate protected residues. Larger arrowheads indicate major protections or hypersensitive sites.

    Journal: Journal of Virology

    Article Title: In Vivo Genomic Footprinting of the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Long Terminal Repeat Enhancer Sequences in HTLV-1-Infected Human T-Cell Lines with Different Levels of Tax I Activity

    doi:

    Figure Lengend Snippet: In vivo DMS footprinting of the lower strand of the TRE-1s. DNAs from in vivo DMS-treated MT-2 and MT-4 cells were piperidine cleaved and subjected to LMPCR as described in Materials and Methods. The reaction products were separated on polyacrylamide gels and exposed to film. Autoradiograms corresponding to the three TRE-1s are shown. (A) Promoter-distal TRE-1. A band is missing in the 5′ GC-rich flank most likely because the proviral DNA contains a base other than the predicted guanine residue at that position. (B) Promoter-central TRE-1. (C) Promoter-proximal TRE-1. Arrowheads pointing toward bands indicate hypersensitive residues; arrowheads pointing away from bands indicate protected residues. Larger arrowheads indicate major protections or hypersensitive sites.

    Article Snippet: This first-strand primer extension was accomplished by incubation of 2 μg of DMS-treated, piperidine-cleaved DNA with 0.3 pmol of oligo 1, 1× Vent DNA polymerase buffer (New England Biolabs), 4 mM MgSO4 , 0.25 mM deoxynucleoside triphosphates (dNTPs), and 0.5 U of Vent DNA polymerase (New England Biolabs) in a total volume of 30 μl.

    Techniques: In Vivo, Footprinting

    Figure 1. Detection of a 100 kD SV40 sT-ag in SV40-transfomed rat cell clones. SDS gel electrophoresis of immunoprecipitated SV40 T-ag-specific proteins from seven (lane 1 to 7) rat 2 cell lines that were transfected with SV40 DNA coding for the

    Journal: RNA Biology

    Article Title: Homologous SV40 RNA trans-splicing

    doi: 10.4161/rna.26707

    Figure Lengend Snippet: Figure 1. Detection of a 100 kD SV40 sT-ag in SV40-transfomed rat cell clones. SDS gel electrophoresis of immunoprecipitated SV40 T-ag-specific proteins from seven (lane 1 to 7) rat 2 cell lines that were transfected with SV40 DNA coding for the

    Article Snippet: PCR was performed as described in standard protocols either with 1/10 of the volume (2 µl) of the cDNA preparation or 1 µg isolated genomic DNA, 0.2 mM of each dNTP, 0.5 µg of a pair of SV40-specific primers, and 1–2 U of Taq DNA polymerase (Perklin Elmer) or proof reading Vent DNA polymerase (NEB) in 100 µl reaction buffer.

    Techniques: Clone Assay, SDS-Gel, Electrophoresis, Immunoprecipitation, Transfection

    Figure 4. Schematic illustration of two mechanisms that can lead to the generation of the sT-ag mRNA and protein. ( A ) Alternative RNA cis -splicing of a long pre-mRNA transcript originating from genomic SV40 DNA tandem integration; this mechanism

    Journal: RNA Biology

    Article Title: Homologous SV40 RNA trans-splicing

    doi: 10.4161/rna.26707

    Figure Lengend Snippet: Figure 4. Schematic illustration of two mechanisms that can lead to the generation of the sT-ag mRNA and protein. ( A ) Alternative RNA cis -splicing of a long pre-mRNA transcript originating from genomic SV40 DNA tandem integration; this mechanism

    Article Snippet: PCR was performed as described in standard protocols either with 1/10 of the volume (2 µl) of the cDNA preparation or 1 µg isolated genomic DNA, 0.2 mM of each dNTP, 0.5 µg of a pair of SV40-specific primers, and 1–2 U of Taq DNA polymerase (Perklin Elmer) or proof reading Vent DNA polymerase (NEB) in 100 µl reaction buffer.

    Techniques:

    Ligation of short padlock probes. Ligation products ( A ) and the Exo III and -VII digestion mixtures ( B ) were separated on 12% denaturing polyacrylamide gels. DNA was revealed using a standard silver-staining method. G and A represent the target genotypes GG and AA from S75 and mutant 109, respectively. M is the 30–330 bp AFLP DNA Ladder (Life Technologies). (A) Linear padlock probes and circularised padlock probes are indicated by solid arrows and asterisks, respectively. (B) After ligation, 5 µl of digestion solution containing 100 U of Exonuclease III and 0.75 U of Exonuclease VII was added to the ligation mixture. Digestion was carried out at 37°C for 45 min and the reaction stopped by heating to 75°C for 10 min. After digestion, only circularised padlock probes derived from the allele-specific ligations remained.

    Journal: Nucleic Acids Research

    Article Title: L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

    doi:

    Figure Lengend Snippet: Ligation of short padlock probes. Ligation products ( A ) and the Exo III and -VII digestion mixtures ( B ) were separated on 12% denaturing polyacrylamide gels. DNA was revealed using a standard silver-staining method. G and A represent the target genotypes GG and AA from S75 and mutant 109, respectively. M is the 30–330 bp AFLP DNA Ladder (Life Technologies). (A) Linear padlock probes and circularised padlock probes are indicated by solid arrows and asterisks, respectively. (B) After ligation, 5 µl of digestion solution containing 100 U of Exonuclease III and 0.75 U of Exonuclease VII was added to the ligation mixture. Digestion was carried out at 37°C for 45 min and the reaction stopped by heating to 75°C for 10 min. After digestion, only circularised padlock probes derived from the allele-specific ligations remained.

    Article Snippet: To circumvent this step, circularised padlock probes were further amplified with one primer using φ29 DNA polymerase (Fig. B) or with two primers using Vent (exo– ) DNA polymerase (Fig. F).

    Techniques: Ligation, Silver Staining, Mutagenesis, Derivative Assay

    Large scale multiplex PCR with 800 primer pairs . Gel electrophoresis of PCR products obtained with high complexity 800-primer pair mix (Additional file 1 ) with a final concentration of 0.02 μM for each individual primer pair and using Taq polymerase (standard LSplex) (A) or using vent exo-polymerase (B and C). Efficiency of LSplex using primer mix with different individual primer concentrations (B). Optimized LSplex amplification of various DNA templates from Gram-negative, Gram-positive bacteria and Candida albicans (C). 100 ng genomic DNA from each indicated species served as template.

    Journal: BMC Microbiology

    Article Title: Large scale multiplex PCR improves pathogen detection by DNA microarrays

    doi: 10.1186/1471-2180-9-1

    Figure Lengend Snippet: Large scale multiplex PCR with 800 primer pairs . Gel electrophoresis of PCR products obtained with high complexity 800-primer pair mix (Additional file 1 ) with a final concentration of 0.02 μM for each individual primer pair and using Taq polymerase (standard LSplex) (A) or using vent exo-polymerase (B and C). Efficiency of LSplex using primer mix with different individual primer concentrations (B). Optimized LSplex amplification of various DNA templates from Gram-negative, Gram-positive bacteria and Candida albicans (C). 100 ng genomic DNA from each indicated species served as template.

    Article Snippet: Reactions in a total volume of 50 μL were performed with 2 U either of Taq DNA polymerase (Fermentas, St. Leon-Rot, Germany) (standard LSplex ) or Vent exo- DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) (optimized LSplex ).

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Concentration Assay, Amplification

    BB0347 is expressed and produced in culture. A) Expression of BB0347 was verified by RT- PCR with flaB mRNA as a control. Lane 1: flaB from genomic DNA, lane 2: flaB from cDNA, lane 3: no RT control, lane 4: bb0347 from genomic DNA, lane 5: bb0347 from cDNA, lane 6: no RT control, L: ladder. B) Western blotting shows that BB0347 protein is produced in the spirochete at all sampled time points. C) QRT- PCR of bb0347 at two different temperatures of incubation with flaB as a standard. D) Western blot using αBB0347 and αFlaB against whole-cell lysates from spirochetes grown at either 34 or 23°C to similar cellular densities. All figures are representative of at least two independent experiments with similar results, and error bars indicate ±SEM.

    Journal: PLoS ONE

    Article Title: BB0347, from the Lyme Disease Spirochete Borrelia burgdorferi, Is Surface Exposed and Interacts with the CS1 Heparin-Binding Domain of Human Fibronectin

    doi: 10.1371/journal.pone.0075643

    Figure Lengend Snippet: BB0347 is expressed and produced in culture. A) Expression of BB0347 was verified by RT- PCR with flaB mRNA as a control. Lane 1: flaB from genomic DNA, lane 2: flaB from cDNA, lane 3: no RT control, lane 4: bb0347 from genomic DNA, lane 5: bb0347 from cDNA, lane 6: no RT control, L: ladder. B) Western blotting shows that BB0347 protein is produced in the spirochete at all sampled time points. C) QRT- PCR of bb0347 at two different temperatures of incubation with flaB as a standard. D) Western blot using αBB0347 and αFlaB against whole-cell lysates from spirochetes grown at either 34 or 23°C to similar cellular densities. All figures are representative of at least two independent experiments with similar results, and error bars indicate ±SEM.

    Article Snippet: Recombinant Protein Production DNA sequences were amplified via PCR using DeepVent DNA Polymerase (NEB, #M0258S; Ipswich, MA) to prevent addition of the 3′ A-overhang onto amplified nucleotide segments.

    Techniques: Produced, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Incubation

    Testing a DNA polymerase with high strand displacement activity in the amplification of the 12 TALE DNA-binding repeats in pTAL2 vector. Various parameters influencing the activity and the outcome of the PCR amplification of the Deep-VentR DNA polymerase are shown. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: Testing a DNA polymerase with high strand displacement activity in the amplification of the 12 TALE DNA-binding repeats in pTAL2 vector. Various parameters influencing the activity and the outcome of the PCR amplification of the Deep-VentR DNA polymerase are shown. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Article Snippet: Deep-VentR DNA polymerase (NE Biolabs) was advertised as the polymerase with one of the highest strand displacement activity and is suitable for thermal cycling.

    Techniques: Activity Assay, Amplification, Binding Assay, Plasmid Preparation, Polymerase Chain Reaction

    ET SSB slightly improves the performance of Deep-VentR DNA polymerase in amplifying TALE DNA-binding repeats. The arrow indicates the expected size of amplification products. ET SSB, a heat resistant single strand DNA-binding protein isolated from thermophilic bacteria (NE Biolabs). PCR conditions are given in the supplementary material .

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: ET SSB slightly improves the performance of Deep-VentR DNA polymerase in amplifying TALE DNA-binding repeats. The arrow indicates the expected size of amplification products. ET SSB, a heat resistant single strand DNA-binding protein isolated from thermophilic bacteria (NE Biolabs). PCR conditions are given in the supplementary material .

    Article Snippet: Deep-VentR DNA polymerase (NE Biolabs) was advertised as the polymerase with one of the highest strand displacement activity and is suitable for thermal cycling.

    Techniques: Binding Assay, Amplification, Isolation, Polymerase Chain Reaction

    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.

    Journal: Nucleic Acids Research

    Article Title: Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs

    doi: 10.1093/nar/gkm017

    Figure Lengend Snippet: Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.

    Article Snippet: For the experiments schematically depicted in , 0.4 µM sense- and antisense MPR primers, four dNTP (0.35 mM each) and 2.6 units of 3′–5′ exo + Vent DNA polymerase (New England Biolabs) were mixed in reaction buffer (10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM Tris-HCl, 2 mM MgSO4 , 0.1% TritonX-100, pH 8.8).

    Techniques: Synthesized, Molecular Weight, Clone Assay