t4 dna polymerase Promega Search Results


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  • 99
    New England Biolabs t4 dna ligase
    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of <t>T4</t> DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher topo ta cloning kit
    (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into <t>pCR</t> <t>II-TOPO</t> vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega t4 dna ligase
    DNA cleavage is coordinated at 3′Dβ and Jβ RSSs. (A) Schematic of the Jβ1 ω , Jβ1 M2 , and Jβ1 M4 alleles as described in the legend to Fig. 1 with the BW linker ligated to cleaved 3′ Dβ1 and Jβ1.2 signal ends. The position of the oligonucleotide primers used to amplify linker ligated to the 3′ Dβ1 (BW-1H, 3A, and 3B) and Jβ1.2 (BWJ, JA, and JB) signal ends are shown as is the position of the oligonucleotide (P1) used to probe these PCR products. Genomic DNA from Jβ1 M4/ω or Jβ1 M2/ω DN thymocytes was incubated with the BW linker in the presence (+) or absence (−) or <t>T4</t> DNA ligase and heminested LMPCRs performed to detect 3′ Dβ1 (B) and Jβ1.2 (C) signal ends as described in the Materials and Methods section. Analyses of Jβ1.2 signal ends was performed on ligated thymocyte DNA that was serially diluted into nonligated DNA keeping the total amount of DNA constant. Cell equivalents of ligated template DNA are indicated. Products from ligation of the BW linker to signal ends from the Jβ1 ω (ω), Jβ1 M4 (M4), and Jβ1 M2 (M2) alleles are indicated. Molecular weight markers are shown. Also shown is a RAG-2 gene PCR performed on ligated and nonligated template DNA and probed with the R2P oligonucleotide.
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 11518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega t4 ligase
    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to <t>T4</t> ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
    T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 2304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega dnase i
    MarR family protein Rv2887 binds to a sequence upstream of the Rv0560c gene. ( A ) EMSA experiment using a PCR-amplified DNA probe spanning the upstream region of Rv0560c. Migration of the DNA probe was retarded compared to free-labeled DNA on addition of Rv2887. A labeled random DNA sequence of the same length as the target probe was used as a control (lane 1) ( B ) Dye primer-based <t>DNase</t> I footprinting shows that Rv2887 binds directly to a sequence upstream of Rv0560c. Electropherograms indicating the protection pattern of the region upstream of Rv0560c on digestion with Dnase I after incubation with (I) 0 μg (II) 0.45 μg or (III) 0.9 μg Rv2887 protein. ( C ) The protected DNA sequence. The DNA sequence upstream of Rv0560c showing the Rv2887 binding site (highlighted in light green).
    Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 14080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega rnase free dnase i
    T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of <t>DNase</t> I and 50 µg/ml of <t>RNase</t> A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.
    Rnase Free Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 5323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i
    Amplification plot for the expression of CD68 expression in the monocytic cells. Oligonucleotide primer pairs for CD68 gene and an oligonucleotide probe labeled with a reporter fluorescent dye at the 5′ end and quencher dye at the 3′ end were designed using Oligo 4.0 software (National Bioscience, Plymouth, MN). Total RNA with <t>DNase</t> I treatment was used to synthesize first-stand cDNA with RT (GIBCOBRL) and oligo(dT) 15 Primer (Promega). Total RNA (50 ng) was added to a 50 μl reverse transcriptase-polymerase chain reaction (RT-PCR) reaction mixture according to the manufacturer’s protocol (Roche Molecular Systems). The products of the RT reactions was used to seed real-time PCR by using an ABI Prism 7700 Sequence Detector by comparing with GAPDH (internal control) and individual standard curve performed in triplicate. The thermal cycling conditions included one cycle at 48°C for 30 minutes, one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 s, annealing at 60°C for 1 minute, and a final hold at 25°C for 2 minutes. Standard curves for the expression of each gene were generated by serial dilution of a standard preparation of total RNA isolated from cultured monocytic or endothelial cells.
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 73125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dnase i
    <t>DNase</t> I disrupts established biofilms of B. bronchiseptica and B. pertussis formed in the mouse respiratory tract. CLSM images of biofilms harvested from mouse nose 15 and 19 days postinoculation with RB50 (top) or Bp536 (bottom), respectively. The harvested nasal septum was excised into two equal parts and incubated either with DNase I buffer (Mock, left panels) or with DNase I (right panels) before processing for staining as described in the Materials and Methods . Green staining depicts Bordetella biofilms formed on top of the host epithelium, which is stained red.
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega t4 dna polymerase
    Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with <t>T4</t> DNA polymerase, which robustly
    T4 Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1056 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase
    Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with <t>T4</t> DNA polymerase, which robustly
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgem t easy vector
    Double digestion of <t>pGEM-T</t> easy vector containing <t>mTEX101</t> fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 91767 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega taq dna polymerase
    Selective and quantitative amplification of targets. ( A ) Selective amplification of <t>DNA</t> flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby <t>Taq</t> I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
    Taq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 19543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pcr clean up system
    Sequence analysis of EZ-Tn5 insertion in the galU mutants. (A) Genomic location of galU on the X. citri subsp. citri chromosome and transposon insertion sites in the galU mutants. kefB encodes a transport protein, galU encodes a UTP-glucose-1-phosphate uridylyltransferase, and XCC2293 encodes a dehydratase protein. Bg, BglII restriction site. (B) <t>PCR</t> analysis confirming insertion of EZ-Tn5 into the galU gene: agarose gel electrophoresis of <t>DNA</t> amplified using primers galU -F1 and galU -R1 targeting the interior region of the galU gene from the X. citri subsp. citri wild-type 306, D12, and F6 strains. Lane 1, Invitrogen 1 Kb Plus DNA size marker; lane 2, D12, lane 3, F6, lane 4, X. citri subsp. citri 306. (C) Southern blot of DNA of X. citri subsp. citri wild-type strain 306 and galU mutants F6 and D12 digested with BglII. The membrane was probed with a 675-bp kan-2 gene fragment that was amplified using PCR with primers Kan-F1 and Kan-R1. Wt, wild type.
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 22690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 ligase
    Joining reactions using mammalian DNA repair proteins. ( A ) Joining reactions using <t>T4</t> ligase (40 units), or DNA ligase IV and XRCC4 (LIV/X4, 250 ng) and Ku heterodimer (Ku, 200 ng), as indicated, were conducted on either intact cleavage products from reactions including MgCl 2 (CP; lanes 1–4) or thoroughly deproteinized cleavage products (dp CP; lanes 5–7). Where indicated, DNA-PK (100 units) was included in the joining reaction. Signal joints (SJ) were detected by PCR. Ethidium bromide-stained gels are shown. ( B ) Joining reactions using thoroughly deproteinized cleavage products were conducted per A with T4 ligase, DNA ligase IV and XRCC4, and/or the Ku heterodimer, as indicated. ( C ) Joining reactions conducted per B were subjected to digestion by Apa LI (5 units) for 60 min at 37°C.
    T4 Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9635 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaquick pcr purification kit
    Joining reactions using mammalian DNA repair proteins. ( A ) Joining reactions using <t>T4</t> ligase (40 units), or DNA ligase IV and XRCC4 (LIV/X4, 250 ng) and Ku heterodimer (Ku, 200 ng), as indicated, were conducted on either intact cleavage products from reactions including MgCl 2 (CP; lanes 1–4) or thoroughly deproteinized cleavage products (dp CP; lanes 5–7). Where indicated, DNA-PK (100 units) was included in the joining reaction. Signal joints (SJ) were detected by PCR. Ethidium bromide-stained gels are shown. ( B ) Joining reactions using thoroughly deproteinized cleavage products were conducted per A with T4 ligase, DNA ligase IV and XRCC4, and/or the Ku heterodimer, as indicated. ( C ) Joining reactions conducted per B were subjected to digestion by Apa LI (5 units) for 60 min at 37°C.
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polynucleotide kinase
    Joining reactions using mammalian DNA repair proteins. ( A ) Joining reactions using <t>T4</t> ligase (40 units), or DNA ligase IV and XRCC4 (LIV/X4, 250 ng) and Ku heterodimer (Ku, 200 ng), as indicated, were conducted on either intact cleavage products from reactions including MgCl 2 (CP; lanes 1–4) or thoroughly deproteinized cleavage products (dp CP; lanes 5–7). Where indicated, DNA-PK (100 units) was included in the joining reaction. Signal joints (SJ) were detected by PCR. Ethidium bromide-stained gels are shown. ( B ) Joining reactions using thoroughly deproteinized cleavage products were conducted per A with T4 ligase, DNA ligase IV and XRCC4, and/or the Ku heterodimer, as indicated. ( C ) Joining reactions conducted per B were subjected to digestion by Apa LI (5 units) for 60 min at 37°C.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 dna polymerase
    Experimental strategy for the analysis of the DNA ends produced by Type I restriction enzymes. Circular plasmid DNA (black lines) containing a single recognition site (gray box) is cleaved by a Type I restriction enzyme (REase) that is represented as one light gray oval (methylase) and two dark gray ovals (HsdR subunits). The linearized DNA is purified from an agarose gel and treated with <t>T4</t> DNA polymerase to remove possible 3′-overhangs or to fill in 5′-overhangs. The resulting blunt-ended DNA fragments are ligated with a fragment carrying a chloramphenicol (Cm) resistance marker (open box) and transformed into bacteria. Plasmid DNA from individual clones is isolated and the sequence of the ends of cloned Type I restriction products is determined using primers originating in the Cm gene. The obtained sequences are aligned with the original pARK sequence. Deletions at the ends of cloned DNA fragments suggest the presence of 3′-overhangs, while sequence duplications indicate 5′-overhangs. No change in the sequence with respect to pARK indicates the presence of blunt ends.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pfu polymerase
    Experimental strategy for the analysis of the DNA ends produced by Type I restriction enzymes. Circular plasmid DNA (black lines) containing a single recognition site (gray box) is cleaved by a Type I restriction enzyme (REase) that is represented as one light gray oval (methylase) and two dark gray ovals (HsdR subunits). The linearized DNA is purified from an agarose gel and treated with <t>T4</t> DNA polymerase to remove possible 3′-overhangs or to fill in 5′-overhangs. The resulting blunt-ended DNA fragments are ligated with a fragment carrying a chloramphenicol (Cm) resistance marker (open box) and transformed into bacteria. Plasmid DNA from individual clones is isolated and the sequence of the ends of cloned Type I restriction products is determined using primers originating in the Cm gene. The obtained sequences are aligned with the original pARK sequence. Deletions at the ends of cloned DNA fragments suggest the presence of 3′-overhangs, while sequence duplications indicate 5′-overhangs. No change in the sequence with respect to pARK indicates the presence of blunt ends.
    Pfu Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 2731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick gel extraction kit
    Experimental strategy for the analysis of the DNA ends produced by Type I restriction enzymes. Circular plasmid DNA (black lines) containing a single recognition site (gray box) is cleaved by a Type I restriction enzyme (REase) that is represented as one light gray oval (methylase) and two dark gray ovals (HsdR subunits). The linearized DNA is purified from an agarose gel and treated with <t>T4</t> DNA polymerase to remove possible 3′-overhangs or to fill in 5′-overhangs. The resulting blunt-ended DNA fragments are ligated with a fragment carrying a chloramphenicol (Cm) resistance marker (open box) and transformed into bacteria. Plasmid DNA from individual clones is isolated and the sequence of the ends of cloned Type I restriction products is determined using primers originating in the Cm gene. The obtained sequences are aligned with the original pARK sequence. Deletions at the ends of cloned DNA fragments suggest the presence of 3′-overhangs, while sequence duplications indicate 5′-overhangs. No change in the sequence with respect to pARK indicates the presence of blunt ends.
    Qiaquick Gel Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 113321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega t4 polynucleotide kinase
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    T4 Polynucleotide Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 5420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa t4 dna ligase
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher clonejet pcr cloning kit
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Clonejet Pcr Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sequence of the <t>pGEM-T-MHBst</t> 167 by direct sequencing.
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    Image Search Results


    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Journal: BMC Molecular Biology

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles

    doi: 10.1186/s12867-017-0083-2

    Figure Lengend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Article Snippet: Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA).

    Techniques: Methylation, Ligation, Polymerase Chain Reaction, Amplification

    (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents

    Journal: Molecular Cancer

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells

    doi: 10.1186/1476-4598-8-67

    Figure Lengend Snippet: (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents "%" distribution.

    Article Snippet: DNA was amplified using the linker as a primer to generate a sufficient amount to clone into the pCR II-TOPO vector using TOPO TA Cloning Kit with One Shot Max Efficiency DH5α-T1 E. coli according to the manufacturer's suggested protocol (Invitrogen).

    Techniques: Chromatin Immunoprecipitation, Clone Assay, Sonication, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Immunoprecipitation, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Genomic Sequencing

    DNA cleavage is coordinated at 3′Dβ and Jβ RSSs. (A) Schematic of the Jβ1 ω , Jβ1 M2 , and Jβ1 M4 alleles as described in the legend to Fig. 1 with the BW linker ligated to cleaved 3′ Dβ1 and Jβ1.2 signal ends. The position of the oligonucleotide primers used to amplify linker ligated to the 3′ Dβ1 (BW-1H, 3A, and 3B) and Jβ1.2 (BWJ, JA, and JB) signal ends are shown as is the position of the oligonucleotide (P1) used to probe these PCR products. Genomic DNA from Jβ1 M4/ω or Jβ1 M2/ω DN thymocytes was incubated with the BW linker in the presence (+) or absence (−) or T4 DNA ligase and heminested LMPCRs performed to detect 3′ Dβ1 (B) and Jβ1.2 (C) signal ends as described in the Materials and Methods section. Analyses of Jβ1.2 signal ends was performed on ligated thymocyte DNA that was serially diluted into nonligated DNA keeping the total amount of DNA constant. Cell equivalents of ligated template DNA are indicated. Products from ligation of the BW linker to signal ends from the Jβ1 ω (ω), Jβ1 M4 (M4), and Jβ1 M2 (M2) alleles are indicated. Molecular weight markers are shown. Also shown is a RAG-2 gene PCR performed on ligated and nonligated template DNA and probed with the R2P oligonucleotide.

    Journal: The Journal of Experimental Medicine

    Article Title: Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

    doi: 10.1084/jem.20011803

    Figure Lengend Snippet: DNA cleavage is coordinated at 3′Dβ and Jβ RSSs. (A) Schematic of the Jβ1 ω , Jβ1 M2 , and Jβ1 M4 alleles as described in the legend to Fig. 1 with the BW linker ligated to cleaved 3′ Dβ1 and Jβ1.2 signal ends. The position of the oligonucleotide primers used to amplify linker ligated to the 3′ Dβ1 (BW-1H, 3A, and 3B) and Jβ1.2 (BWJ, JA, and JB) signal ends are shown as is the position of the oligonucleotide (P1) used to probe these PCR products. Genomic DNA from Jβ1 M4/ω or Jβ1 M2/ω DN thymocytes was incubated with the BW linker in the presence (+) or absence (−) or T4 DNA ligase and heminested LMPCRs performed to detect 3′ Dβ1 (B) and Jβ1.2 (C) signal ends as described in the Materials and Methods section. Analyses of Jβ1.2 signal ends was performed on ligated thymocyte DNA that was serially diluted into nonligated DNA keeping the total amount of DNA constant. Cell equivalents of ligated template DNA are indicated. Products from ligation of the BW linker to signal ends from the Jβ1 ω (ω), Jβ1 M4 (M4), and Jβ1 M2 (M2) alleles are indicated. Molecular weight markers are shown. Also shown is a RAG-2 gene PCR performed on ligated and nonligated template DNA and probed with the R2P oligonucleotide.

    Article Snippet: Purified thymocyte genomic DNA (2–3 μg) was ligated to 100 pmoles of the BW linker in a volume of 50–60 μl with 1–3 units T4 DNA Ligase (Promega) at 16°C for 12–14 h. Ligated samples were extracted with phenol and chloroform before PCR analysis.

    Techniques: Polymerase Chain Reaction, Incubation, Ligation, Molecular Weight

    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

    Journal: Journal of Biological Engineering

    Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    doi: 10.1186/1754-1611-1-7

    Figure Lengend Snippet: Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

    Article Snippet: Enzyme amounts: 7.5 units Esp3I, 10 units NheI, 3 weiss units T4 ligase.

    Techniques: Subcloning, Plasmid Preparation, Activity Assay, Marker

    MarR family protein Rv2887 binds to a sequence upstream of the Rv0560c gene. ( A ) EMSA experiment using a PCR-amplified DNA probe spanning the upstream region of Rv0560c. Migration of the DNA probe was retarded compared to free-labeled DNA on addition of Rv2887. A labeled random DNA sequence of the same length as the target probe was used as a control (lane 1) ( B ) Dye primer-based DNase I footprinting shows that Rv2887 binds directly to a sequence upstream of Rv0560c. Electropherograms indicating the protection pattern of the region upstream of Rv0560c on digestion with Dnase I after incubation with (I) 0 μg (II) 0.45 μg or (III) 0.9 μg Rv2887 protein. ( C ) The protected DNA sequence. The DNA sequence upstream of Rv0560c showing the Rv2887 binding site (highlighted in light green).

    Journal: Scientific Reports

    Article Title: Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis

    doi: 10.1038/s41598-017-01705-4

    Figure Lengend Snippet: MarR family protein Rv2887 binds to a sequence upstream of the Rv0560c gene. ( A ) EMSA experiment using a PCR-amplified DNA probe spanning the upstream region of Rv0560c. Migration of the DNA probe was retarded compared to free-labeled DNA on addition of Rv2887. A labeled random DNA sequence of the same length as the target probe was used as a control (lane 1) ( B ) Dye primer-based DNase I footprinting shows that Rv2887 binds directly to a sequence upstream of Rv0560c. Electropherograms indicating the protection pattern of the region upstream of Rv0560c on digestion with Dnase I after incubation with (I) 0 μg (II) 0.45 μg or (III) 0.9 μg Rv2887 protein. ( C ) The protected DNA sequence. The DNA sequence upstream of Rv0560c showing the Rv2887 binding site (highlighted in light green).

    Article Snippet: After incubation for 30 min at 25 °C, 10 µl solution containing about 0.015 units DNase I (Promega) and 100 nM freshly prepared CaCl2 was added and further incubated for 1 min at 25 °C.

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification, Migration, Labeling, Footprinting, Incubation, Binding Assay

    T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

    Journal: Nucleic Acids Research

    Article Title: Transformation of isolated mammalian mitochondria by bacterial conjugation

    doi: 10.1093/nar/gni140

    Figure Lengend Snippet: T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

    Article Snippet: RT–PCR analysis Total RNAs from the T7RNAP-mitochondria mating mixture or from electroporated mitochondria were collected by isopropyl alcohol precipitation, and residual DNA contaminants were removed using RNase-free DNase I (Promega, Madison, WI) as directed by the manufacturer.

    Techniques: Conjugation Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

    Amplification plot for the expression of CD68 expression in the monocytic cells. Oligonucleotide primer pairs for CD68 gene and an oligonucleotide probe labeled with a reporter fluorescent dye at the 5′ end and quencher dye at the 3′ end were designed using Oligo 4.0 software (National Bioscience, Plymouth, MN). Total RNA with DNase I treatment was used to synthesize first-stand cDNA with RT (GIBCOBRL) and oligo(dT) 15 Primer (Promega). Total RNA (50 ng) was added to a 50 μl reverse transcriptase-polymerase chain reaction (RT-PCR) reaction mixture according to the manufacturer’s protocol (Roche Molecular Systems). The products of the RT reactions was used to seed real-time PCR by using an ABI Prism 7700 Sequence Detector by comparing with GAPDH (internal control) and individual standard curve performed in triplicate. The thermal cycling conditions included one cycle at 48°C for 30 minutes, one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 s, annealing at 60°C for 1 minute, and a final hold at 25°C for 2 minutes. Standard curves for the expression of each gene were generated by serial dilution of a standard preparation of total RNA isolated from cultured monocytic or endothelial cells.

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    Article Title: Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells

    doi: 10.1161/01.ATV.0000222017.05085.8e

    Figure Lengend Snippet: Amplification plot for the expression of CD68 expression in the monocytic cells. Oligonucleotide primer pairs for CD68 gene and an oligonucleotide probe labeled with a reporter fluorescent dye at the 5′ end and quencher dye at the 3′ end were designed using Oligo 4.0 software (National Bioscience, Plymouth, MN). Total RNA with DNase I treatment was used to synthesize first-stand cDNA with RT (GIBCOBRL) and oligo(dT) 15 Primer (Promega). Total RNA (50 ng) was added to a 50 μl reverse transcriptase-polymerase chain reaction (RT-PCR) reaction mixture according to the manufacturer’s protocol (Roche Molecular Systems). The products of the RT reactions was used to seed real-time PCR by using an ABI Prism 7700 Sequence Detector by comparing with GAPDH (internal control) and individual standard curve performed in triplicate. The thermal cycling conditions included one cycle at 48°C for 30 minutes, one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 s, annealing at 60°C for 1 minute, and a final hold at 25°C for 2 minutes. Standard curves for the expression of each gene were generated by serial dilution of a standard preparation of total RNA isolated from cultured monocytic or endothelial cells.

    Article Snippet: Extracted RNA was treated with DNase I (Ambion) in the presence of RNasin (Promega) to remove DNA contamination before cDNA synthesis. cDNA was synthesized with oligodeoxythymidylic acid primers (Boehringer Mannheim) and Superscript 11 reverse transcriptase (Life Technologies, Inc.).

    Techniques: Amplification, Expressing, Labeling, Software, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Generated, Serial Dilution, Isolation, Cell Culture

    DNase I disrupts established biofilms of B. bronchiseptica and B. pertussis formed in the mouse respiratory tract. CLSM images of biofilms harvested from mouse nose 15 and 19 days postinoculation with RB50 (top) or Bp536 (bottom), respectively. The harvested nasal septum was excised into two equal parts and incubated either with DNase I buffer (Mock, left panels) or with DNase I (right panels) before processing for staining as described in the Materials and Methods . Green staining depicts Bordetella biofilms formed on top of the host epithelium, which is stained red.

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I disrupts established biofilms of B. bronchiseptica and B. pertussis formed in the mouse respiratory tract. CLSM images of biofilms harvested from mouse nose 15 and 19 days postinoculation with RB50 (top) or Bp536 (bottom), respectively. The harvested nasal septum was excised into two equal parts and incubated either with DNase I buffer (Mock, left panels) or with DNase I (right panels) before processing for staining as described in the Materials and Methods . Green staining depicts Bordetella biofilms formed on top of the host epithelium, which is stained red.

    Article Snippet: DNase I from Sigma or from Promega was incubated at 37°C for 48 h in the reaction buffer.

    Techniques: Confocal Laser Scanning Microscopy, Incubation, Staining

    Scanning electron microscopy of mock treated (left) or DNase I treated Bordetella biofilms formed in the mouse nose. Nasal septa were harvested from mice 15 days post-inoculation, excised into two equal parts, treated with either the DNase I buffer (Mock, left panels) or DNase I (right) followed by processing for SEM as described in the Materials and Methods .

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: Scanning electron microscopy of mock treated (left) or DNase I treated Bordetella biofilms formed in the mouse nose. Nasal septa were harvested from mice 15 days post-inoculation, excised into two equal parts, treated with either the DNase I buffer (Mock, left panels) or DNase I (right) followed by processing for SEM as described in the Materials and Methods .

    Article Snippet: DNase I from Sigma or from Promega was incubated at 37°C for 48 h in the reaction buffer.

    Techniques: Electron Microscopy, Mouse Assay

    DNase I leads to the disruption of established Bordetella biofilms grown in microtitre plates. Preformed 48h RB50 biofilms grown in 96 well plates were rinsed with PBS followed by incubation with PBS, PBS and reaction buffer, PBS and DNase I, PBS and heat inactivated (HI) DNase I, or PBS with reaction buffer and DNase I. Biofilm formation was then quantitated via crystal violet staining. Each point is an average of at least 6 wells, and error bars indicate the standard deviation. Asterisks designate a value of P

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I leads to the disruption of established Bordetella biofilms grown in microtitre plates. Preformed 48h RB50 biofilms grown in 96 well plates were rinsed with PBS followed by incubation with PBS, PBS and reaction buffer, PBS and DNase I, PBS and heat inactivated (HI) DNase I, or PBS with reaction buffer and DNase I. Biofilm formation was then quantitated via crystal violet staining. Each point is an average of at least 6 wells, and error bars indicate the standard deviation. Asterisks designate a value of P

    Article Snippet: DNase I from Sigma or from Promega was incubated at 37°C for 48 h in the reaction buffer.

    Techniques: Incubation, Staining, Standard Deviation

    DNase I inhibits Bordetella biofilm formation. The indicated strains were grown in 96 well microtitre plates for RB50 or 12 well plates for Bp536 for designated time points in SS medium supplemented with either DNase I resuspended in the reaction buffer or the reaction buffer alone. Wells were rinsed and stained with crystal violet followed by quantification of bound crystal violet as described in Materials and Methods . Each data point is the average of six wells, and error bars indicate the standard deviation. Representative data from one of at least three independent experiments are shown. Asterisks designate a value of P

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I inhibits Bordetella biofilm formation. The indicated strains were grown in 96 well microtitre plates for RB50 or 12 well plates for Bp536 for designated time points in SS medium supplemented with either DNase I resuspended in the reaction buffer or the reaction buffer alone. Wells were rinsed and stained with crystal violet followed by quantification of bound crystal violet as described in Materials and Methods . Each data point is the average of six wells, and error bars indicate the standard deviation. Representative data from one of at least three independent experiments are shown. Asterisks designate a value of P

    Article Snippet: DNase I from Sigma or from Promega was incubated at 37°C for 48 h in the reaction buffer.

    Techniques: Staining, Standard Deviation

    Susceptibility of flow cell biofilms to DNase I. Representative z-reconstructions of RB50 biofilms grown under flow conditions for 6, 72, or 120h and imaged using CLSM for live GFP expressing cells (green) and eDNA stained with DDAO (red or yellow with co-localization). The image of untreated biofilms (left panels) were taken immediately prior to incubation with DNase I and the images of same biofilms treated with DNase I for 1.5h (left panels). Images shown here are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: Susceptibility of flow cell biofilms to DNase I. Representative z-reconstructions of RB50 biofilms grown under flow conditions for 6, 72, or 120h and imaged using CLSM for live GFP expressing cells (green) and eDNA stained with DDAO (red or yellow with co-localization). The image of untreated biofilms (left panels) were taken immediately prior to incubation with DNase I and the images of same biofilms treated with DNase I for 1.5h (left panels). Images shown here are representative of two independent experiments.

    Article Snippet: DNase I from Sigma or from Promega was incubated at 37°C for 48 h in the reaction buffer.

    Techniques: Flow Cytometry, Confocal Laser Scanning Microscopy, Expressing, Staining, Incubation

    DNase I leads to the disruption of established Bordetella biofilms grown on glass coverslips under static conditions. Biofilms were grown on glass coverslips for 48h for RB50 (A) and 96 h for Bp536 (B). The coverslips were gently rinsed followed by treatment with DNase I for either 30min or 90min. The cells were tagged with GFP and thus are green. For each micrograph, the middle panel represents the x-y plane, and the adjacent top and side panels represent the x-z and y-z planes, respectively. The images of a biofilm not treated with DNase I and treated only with DNase I buffer are also depicted. CLSM was utilized to image the biofilms.

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I leads to the disruption of established Bordetella biofilms grown on glass coverslips under static conditions. Biofilms were grown on glass coverslips for 48h for RB50 (A) and 96 h for Bp536 (B). The coverslips were gently rinsed followed by treatment with DNase I for either 30min or 90min. The cells were tagged with GFP and thus are green. For each micrograph, the middle panel represents the x-y plane, and the adjacent top and side panels represent the x-z and y-z planes, respectively. The images of a biofilm not treated with DNase I and treated only with DNase I buffer are also depicted. CLSM was utilized to image the biofilms.

    Article Snippet: DNase I from Sigma or from Promega was incubated at 37°C for 48 h in the reaction buffer.

    Techniques: Confocal Laser Scanning Microscopy

    Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with T4 DNA polymerase, which robustly

    Journal: The Journal of Biological Chemistry

    Article Title: The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex *

    doi: 10.1074/jbc.M114.570333

    Figure Lengend Snippet: Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with T4 DNA polymerase, which robustly

    Article Snippet: T4 DNA polymerase (Promega Corporation; stock concentration 25 n m ) was used as a control for the primer extensions on hetero-DNA and RNA.

    Techniques:

    Double digestion of pGEM-T easy vector containing mTEX101 fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.

    Journal: Journal of Reproduction & Infertility

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein

    doi:

    Figure Lengend Snippet: Double digestion of pGEM-T easy vector containing mTEX101 fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.

    Article Snippet: Cloning of mTEX101 in TA vector The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product.

    Techniques: Plasmid Preparation

    Coloy PCR on transformed JM109 clones by pGEM-T Easy vector carrying mTEX101 gene. 1-5: 5 selected white colonies, 6: negative control (blue colony), 7: positive control (PCR product on testis cDNA), 8: DNA ladder VIII (Roche).

    Journal: Journal of Reproduction & Infertility

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein

    doi:

    Figure Lengend Snippet: Coloy PCR on transformed JM109 clones by pGEM-T Easy vector carrying mTEX101 gene. 1-5: 5 selected white colonies, 6: negative control (blue colony), 7: positive control (PCR product on testis cDNA), 8: DNA ladder VIII (Roche).

    Article Snippet: Cloning of mTEX101 in TA vector The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product.

    Techniques: Polymerase Chain Reaction, Transformation Assay, Clone Assay, Plasmid Preparation, Negative Control, Positive Control

    Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

    Journal: Nucleic Acids Research

    Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

    doi:

    Figure Lengend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

    Article Snippet: A typical 10 µl ligation reaction contained 250 ng of DNA fragments, 5 µM Taq I adapters, 25 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 5 mM dithiothreitol, 500 µΜ ATP, 12.5 µg/ml BSA and 200 cohesive-end units of T4 DNA ligase (New England Biolabs) and was incubated at 16°C for 12–16 h. Primary PCR was done in a 25 µl reaction volume containing 200 µM each dNTP, 40 nM each primer (Taq1-N 5′-ATGAGTCCTGACCGA, and Tn3-O2, 5′-TTAACGTGAGTTTTCGTTCCACTG), 2 mM MgSO4 and 1 U Taq DNA polymerase (Promega) in 1× PCR buffer (20 mM Tris–HCl pH 9.2, 10 mM KCl, 10 mM ammonium sulfate, 0.1% Triton X-100) and adapter-ligated DNA.

    Techniques: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

    Sequence analysis of EZ-Tn5 insertion in the galU mutants. (A) Genomic location of galU on the X. citri subsp. citri chromosome and transposon insertion sites in the galU mutants. kefB encodes a transport protein, galU encodes a UTP-glucose-1-phosphate uridylyltransferase, and XCC2293 encodes a dehydratase protein. Bg, BglII restriction site. (B) PCR analysis confirming insertion of EZ-Tn5 into the galU gene: agarose gel electrophoresis of DNA amplified using primers galU -F1 and galU -R1 targeting the interior region of the galU gene from the X. citri subsp. citri wild-type 306, D12, and F6 strains. Lane 1, Invitrogen 1 Kb Plus DNA size marker; lane 2, D12, lane 3, F6, lane 4, X. citri subsp. citri 306. (C) Southern blot of DNA of X. citri subsp. citri wild-type strain 306 and galU mutants F6 and D12 digested with BglII. The membrane was probed with a 675-bp kan-2 gene fragment that was amplified using PCR with primers Kan-F1 and Kan-R1. Wt, wild type.

    Journal: Applied and Environmental Microbiology

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri ▿

    doi: 10.1128/AEM.02897-09

    Figure Lengend Snippet: Sequence analysis of EZ-Tn5 insertion in the galU mutants. (A) Genomic location of galU on the X. citri subsp. citri chromosome and transposon insertion sites in the galU mutants. kefB encodes a transport protein, galU encodes a UTP-glucose-1-phosphate uridylyltransferase, and XCC2293 encodes a dehydratase protein. Bg, BglII restriction site. (B) PCR analysis confirming insertion of EZ-Tn5 into the galU gene: agarose gel electrophoresis of DNA amplified using primers galU -F1 and galU -R1 targeting the interior region of the galU gene from the X. citri subsp. citri wild-type 306, D12, and F6 strains. Lane 1, Invitrogen 1 Kb Plus DNA size marker; lane 2, D12, lane 3, F6, lane 4, X. citri subsp. citri 306. (C) Southern blot of DNA of X. citri subsp. citri wild-type strain 306 and galU mutants F6 and D12 digested with BglII. The membrane was probed with a 675-bp kan-2 gene fragment that was amplified using PCR with primers Kan-F1 and Kan-R1. Wt, wild type.

    Article Snippet: The digested DNA was purified using the Wizard SV gel and PCR clean-up system (Promega, Madison, WI) and allowed to self-ligate in the presence of T4 DNA ligase in a 10-μl mixture for 4 h at 16°C.

    Techniques: Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Marker, Southern Blot

    Joining reactions using mammalian DNA repair proteins. ( A ) Joining reactions using T4 ligase (40 units), or DNA ligase IV and XRCC4 (LIV/X4, 250 ng) and Ku heterodimer (Ku, 200 ng), as indicated, were conducted on either intact cleavage products from reactions including MgCl 2 (CP; lanes 1–4) or thoroughly deproteinized cleavage products (dp CP; lanes 5–7). Where indicated, DNA-PK (100 units) was included in the joining reaction. Signal joints (SJ) were detected by PCR. Ethidium bromide-stained gels are shown. ( B ) Joining reactions using thoroughly deproteinized cleavage products were conducted per A with T4 ligase, DNA ligase IV and XRCC4, and/or the Ku heterodimer, as indicated. ( C ) Joining reactions conducted per B were subjected to digestion by Apa LI (5 units) for 60 min at 37°C.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Intermediates in V(D)J recombination: A stable RAG1/2 complex sequesters cleaved RSS ends

    doi: 10.1073/pnas.221471198

    Figure Lengend Snippet: Joining reactions using mammalian DNA repair proteins. ( A ) Joining reactions using T4 ligase (40 units), or DNA ligase IV and XRCC4 (LIV/X4, 250 ng) and Ku heterodimer (Ku, 200 ng), as indicated, were conducted on either intact cleavage products from reactions including MgCl 2 (CP; lanes 1–4) or thoroughly deproteinized cleavage products (dp CP; lanes 5–7). Where indicated, DNA-PK (100 units) was included in the joining reaction. Signal joints (SJ) were detected by PCR. Ethidium bromide-stained gels are shown. ( B ) Joining reactions using thoroughly deproteinized cleavage products were conducted per A with T4 ligase, DNA ligase IV and XRCC4, and/or the Ku heterodimer, as indicated. ( C ) Joining reactions conducted per B were subjected to digestion by Apa LI (5 units) for 60 min at 37°C.

    Article Snippet: T4 ligase, T4 polynucleotide kinase, and all restriction enzymes were purchased from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Staining

    Experimental strategy for the analysis of the DNA ends produced by Type I restriction enzymes. Circular plasmid DNA (black lines) containing a single recognition site (gray box) is cleaved by a Type I restriction enzyme (REase) that is represented as one light gray oval (methylase) and two dark gray ovals (HsdR subunits). The linearized DNA is purified from an agarose gel and treated with T4 DNA polymerase to remove possible 3′-overhangs or to fill in 5′-overhangs. The resulting blunt-ended DNA fragments are ligated with a fragment carrying a chloramphenicol (Cm) resistance marker (open box) and transformed into bacteria. Plasmid DNA from individual clones is isolated and the sequence of the ends of cloned Type I restriction products is determined using primers originating in the Cm gene. The obtained sequences are aligned with the original pARK sequence. Deletions at the ends of cloned DNA fragments suggest the presence of 3′-overhangs, while sequence duplications indicate 5′-overhangs. No change in the sequence with respect to pARK indicates the presence of blunt ends.

    Journal: Nucleic Acids Research

    Article Title: On the DNA cleavage mechanism of Type I restriction enzymes

    doi: 10.1093/nar/gki322

    Figure Lengend Snippet: Experimental strategy for the analysis of the DNA ends produced by Type I restriction enzymes. Circular plasmid DNA (black lines) containing a single recognition site (gray box) is cleaved by a Type I restriction enzyme (REase) that is represented as one light gray oval (methylase) and two dark gray ovals (HsdR subunits). The linearized DNA is purified from an agarose gel and treated with T4 DNA polymerase to remove possible 3′-overhangs or to fill in 5′-overhangs. The resulting blunt-ended DNA fragments are ligated with a fragment carrying a chloramphenicol (Cm) resistance marker (open box) and transformed into bacteria. Plasmid DNA from individual clones is isolated and the sequence of the ends of cloned Type I restriction products is determined using primers originating in the Cm gene. The obtained sequences are aligned with the original pARK sequence. Deletions at the ends of cloned DNA fragments suggest the presence of 3′-overhangs, while sequence duplications indicate 5′-overhangs. No change in the sequence with respect to pARK indicates the presence of blunt ends.

    Article Snippet: The linear products from the cleavage reactions of EcoKI, EcoAI and EcoR124I with pARK were purified from agarose gel using a gel extraction kit (Qiagen) and blunt-ended by incubation with T4 DNA polymerase (NEB) in the presence of all four dNTPs.

    Techniques: Produced, Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Marker, Transformation Assay, Clone Assay, Isolation, Sequencing

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Journal: Nature biotechnology

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    doi: 10.1038/nbt736

    Figure Lengend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Article Snippet: Restriction endonucleases, T4 DNA ligase, Vent DNA polymerase, T4 polynucleotide kinase, and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA).

    Techniques: Ligation, Binding Assay

    Sequence of the pGEM-T-MHBst 167 by direct sequencing.

    Journal: Molecular Medicine Reports

    Article Title: Screening of hepatocyte proteins binding with C-terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two-hybrid system

    doi: 10.3892/mmr.2014.2356

    Figure Lengend Snippet: Sequence of the pGEM-T-MHBst 167 by direct sequencing.

    Article Snippet: Chemical agents and culture media The pGEM-T vector and Taq DNA polymerase were purchased from Promega Corp. (Madison, WI, USA).

    Techniques: Sequencing

    Construction and identification of plasmids by agarose gel electrophoresis. (A) Polymerase chain reaction product MHBst 167 (501 bp). (B) Restriction enzyme digestion of pGBKT7-MHBst 167 plasmids by Eco RI/ Bam HI (pGBKT7, 7,300 bp; MHBst 167 , 501 bp). (C) Restriction enzyme digestion of (Lane 1) pGBKT7-MHBst 167 and (Lane 2) pGADT7-MHBst 167 by Eco RI/ Bam HI. (D) Restriction enzyme digestion of pGEM-T-MHBst 167 plasmids by Eco RI/ Bam HI. M, DL2000 DNA marker; bp, base pairs.

    Journal: Molecular Medicine Reports

    Article Title: Screening of hepatocyte proteins binding with C-terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two-hybrid system

    doi: 10.3892/mmr.2014.2356

    Figure Lengend Snippet: Construction and identification of plasmids by agarose gel electrophoresis. (A) Polymerase chain reaction product MHBst 167 (501 bp). (B) Restriction enzyme digestion of pGBKT7-MHBst 167 plasmids by Eco RI/ Bam HI (pGBKT7, 7,300 bp; MHBst 167 , 501 bp). (C) Restriction enzyme digestion of (Lane 1) pGBKT7-MHBst 167 and (Lane 2) pGADT7-MHBst 167 by Eco RI/ Bam HI. (D) Restriction enzyme digestion of pGEM-T-MHBst 167 plasmids by Eco RI/ Bam HI. M, DL2000 DNA marker; bp, base pairs.

    Article Snippet: Chemical agents and culture media The pGEM-T vector and Taq DNA polymerase were purchased from Promega Corp. (Madison, WI, USA).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker