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  • 99
    New England Biolabs klenow fragment
    Schematic overview of random deletion used in this study . A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and <t>Klenow</t> fragment. D) Single digested with Bgl II. E) Random ligation with T4 <t>DNA</t> ligase.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna polymerase i
    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear <t>DNA</t> using DNA <t>polymerase</t> I and Ampligase. This process substantially improved the complexity of the library during sequencing.
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs klenow dna polymerase
    The principle of PE-PCR for bacterial <t>DNA</t> amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and <t>Klenow</t> DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
    Klenow Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa klenow fragment
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Klenow Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega klenow fragment
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher klenow dna polymerase
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc klenow fragment
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Fragment, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher klenow polymerase
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher klenow
    Rv1222 does not inhibit DNA synthesis. (A) In vitro replication assay: 0.5U of Ec <t>Klenow</t> DNAP, Cy5-labelled primer and 65 base DNA fragments were used in the assay. Rv1222 was incubated with DNAP before the replication reactions were initiated with <t>dNTP.</t> Reactions were stopped after 1min and products were separated on 12% Urea-PAGE. The gel was scanned on a Typhoon Trio+ at Cy5 channel. ( B ) Binding of 20 nM TMR labelled Rv1222 to Klenow polymerase. ( C ) Effect of Rv1222 on the kinetics of DNA replication: Same as A except the reactions were stopped at the indicated time interval. ( D ) In vitro transcription assay with T7 RNAP: 0.2 U of T7 RNAP was used with 100 nM of T7 promoter containing DNA fragment. ( E ) Binding of 20 nM TMR labelled Rv1222 to T7 RNAP by fluorescence anisotropy.
    Klenow, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc klenow exo
    Rv1222 does not inhibit DNA synthesis. (A) In vitro replication assay: 0.5U of Ec <t>Klenow</t> DNAP, Cy5-labelled primer and 65 base DNA fragments were used in the assay. Rv1222 was incubated with DNAP before the replication reactions were initiated with <t>dNTP.</t> Reactions were stopped after 1min and products were separated on 12% Urea-PAGE. The gel was scanned on a Typhoon Trio+ at Cy5 channel. ( B ) Binding of 20 nM TMR labelled Rv1222 to Klenow polymerase. ( C ) Effect of Rv1222 on the kinetics of DNA replication: Same as A except the reactions were stopped at the indicated time interval. ( D ) In vitro transcription assay with T7 RNAP: 0.2 U of T7 RNAP was used with 100 nM of T7 promoter containing DNA fragment. ( E ) Binding of 20 nM TMR labelled Rv1222 to T7 RNAP by fluorescence anisotropy.
    Klenow Exo, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Fragment, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher klenow enzyme
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega large klenow fragment
    Increasing efficiency of detection of low MW restriction fragments. Using <t>Klenow</t> <t>DNA</t> polymerase and a probe with a sub-family specific 3' end and 10x G residues at the 5' end, the efficiency of the labelling reaction could be increased ten fold. Degenerate sub-family centred PCR was performed using the Kir 2.0 subclones as template, labelled as described in Materials and Methods, and digested with Hinf I. Low molecular weight fragment (
    Large Klenow Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzymatics klenow exo
    Increasing efficiency of detection of low MW restriction fragments. Using <t>Klenow</t> <t>DNA</t> polymerase and a probe with a sub-family specific 3' end and 10x G residues at the 5' end, the efficiency of the labelling reaction could be increased ten fold. Degenerate sub-family centred PCR was performed using the Kir 2.0 subclones as template, labelled as described in Materials and Methods, and digested with Hinf I. Low molecular weight fragment (
    Klenow Exo, supplied by Enzymatics, used in various techniques. Bioz Stars score: 92/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene klenow fragment
    Increasing efficiency of detection of low MW restriction fragments. Using <t>Klenow</t> <t>DNA</t> polymerase and a probe with a sub-family specific 3' end and 10x G residues at the 5' end, the efficiency of the labelling reaction could be increased ten fold. Degenerate sub-family centred PCR was performed using the Kir 2.0 subclones as template, labelled as described in Materials and Methods, and digested with Hinf I. Low molecular weight fragment (
    Klenow Fragment, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega klenow enzyme
    Increasing efficiency of detection of low MW restriction fragments. Using <t>Klenow</t> <t>DNA</t> polymerase and a probe with a sub-family specific 3' end and 10x G residues at the 5' end, the efficiency of the labelling reaction could be increased ten fold. Degenerate sub-family centred PCR was performed using the Kir 2.0 subclones as template, labelled as described in Materials and Methods, and digested with Hinf I. Low molecular weight fragment (
    Klenow Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Schematic overview of random deletion used in this study . A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and Klenow fragment. D) Single digested with Bgl II. E) Random ligation with T4 DNA ligase.

    Journal: Microbial Cell Factories

    Article Title: An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

    doi: 10.1186/1475-2859-8-66

    Figure Lengend Snippet: Schematic overview of random deletion used in this study . A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and Klenow fragment. D) Single digested with Bgl II. E) Random ligation with T4 DNA ligase.

    Article Snippet: To facilitate the re-ligation (see below), the purified DNA was blunt-ended with 3.2 U Klenow fragment in 10× NEBuffer 2 (New England Biolabs) and dNTPs (final concentration 33 μM each nucleotide) for 15 min at 25°C.

    Techniques: Plasmid Preparation, Ligation

    A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Journal: Human Genomics

    Article Title: Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

    doi: 10.1186/s40246-018-0160-8

    Figure Lengend Snippet: A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Article Snippet: DNA Polymerase I Large (Klenow) Fragment, restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs (NEB), USA. dNTP mix was purchased from Promega, USA.

    Techniques: Ligation

    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the Klenow fragment exo (−).

    Journal: Nucleic Acids Research

    Article Title: Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA

    doi: 10.1093/nar/gky452

    Figure Lengend Snippet: Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the Klenow fragment exo (−).

    Article Snippet: The reaction was supplemented with 1 μl (20 units) of Exonuclease I (New England BioLabs) and incubated at 37°C for 15 min. Then, the reaction was supplemented with 1 μl (50 units) of Klenow fragment exo (−) (New England BioLabs) and further incubated for 15 min at 37°C.

    Techniques: Primer Extension Assay

    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Journal: Nature biotechnology

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

    doi: 10.1038/nbt.2720

    Figure Lengend Snippet: Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Article Snippet: 10 U of DNA Polymerase I (Invitrogen, Carlsbad, CA) was added to the denatured amplicons along with 250 nanograms of unmodified random hexamer primer, 1 mM dNTPs, 1x Ampligase buffer (Epicentre, Madison, Wi), and 1x NEB buffer 2 (NEB, Cambridge, MA).

    Techniques: Amplification, Lysis, Neutralization, Multiple Displacement Amplification, Transferring, Microscopy, Fluorescence, Micromanipulation, Electron Microscopy, Incubation, Evaporation, Sequencing

    The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Journal: PLoS ONE

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    doi: 10.1371/journal.pone.0020303

    Figure Lengend Snippet: The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Article Snippet: Materials The exo I and Klenow DNA polymerase were purchased from New England Biolab (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Incubation, BAC Assay

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Journal: Nature

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    doi: 10.1038/nature25449

    Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

    Techniques: Inhibition, Immunofluorescence, Staining

    The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

    doi: 10.1093/nar/gkh192

    Figure Lengend Snippet: The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Article Snippet: A fragment containing MxA gene was prepared from pET14b-MxA by digestion with NdeI followed by treatment with Klenow fragment and digestion with BamHI.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Expressing, Amplification, Polymerase Chain Reaction, Derivative Assay, Construct, Clone Assay

    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration

    Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: End Labeling, Labeling

    Rv1222 does not inhibit DNA synthesis. (A) In vitro replication assay: 0.5U of Ec Klenow DNAP, Cy5-labelled primer and 65 base DNA fragments were used in the assay. Rv1222 was incubated with DNAP before the replication reactions were initiated with dNTP. Reactions were stopped after 1min and products were separated on 12% Urea-PAGE. The gel was scanned on a Typhoon Trio+ at Cy5 channel. ( B ) Binding of 20 nM TMR labelled Rv1222 to Klenow polymerase. ( C ) Effect of Rv1222 on the kinetics of DNA replication: Same as A except the reactions were stopped at the indicated time interval. ( D ) In vitro transcription assay with T7 RNAP: 0.2 U of T7 RNAP was used with 100 nM of T7 promoter containing DNA fragment. ( E ) Binding of 20 nM TMR labelled Rv1222 to T7 RNAP by fluorescence anisotropy.

    Journal: Nucleic Acids Research

    Article Title: Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA

    doi: 10.1093/nar/gkv516

    Figure Lengend Snippet: Rv1222 does not inhibit DNA synthesis. (A) In vitro replication assay: 0.5U of Ec Klenow DNAP, Cy5-labelled primer and 65 base DNA fragments were used in the assay. Rv1222 was incubated with DNAP before the replication reactions were initiated with dNTP. Reactions were stopped after 1min and products were separated on 12% Urea-PAGE. The gel was scanned on a Typhoon Trio+ at Cy5 channel. ( B ) Binding of 20 nM TMR labelled Rv1222 to Klenow polymerase. ( C ) Effect of Rv1222 on the kinetics of DNA replication: Same as A except the reactions were stopped at the indicated time interval. ( D ) In vitro transcription assay with T7 RNAP: 0.2 U of T7 RNAP was used with 100 nM of T7 promoter containing DNA fragment. ( E ) Binding of 20 nM TMR labelled Rv1222 to T7 RNAP by fluorescence anisotropy.

    Article Snippet: 0.5 U of Klenow (Thermo Scientific) and 0.25 mM dNTP mix were added to the sample and incubation was carried on for another 1 min ( ).

    Techniques: DNA Synthesis, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Fluorescence

    The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Journal: Nucleic Acids Research

    Article Title: A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

    doi:

    Figure Lengend Snippet: The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Article Snippet: Exonuclease III (exo III) and the Klenow fragment of Escherichia coli were purchased from Boehringer Mannheim and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation, Labeling

    Increasing efficiency of detection of low MW restriction fragments. Using Klenow DNA polymerase and a probe with a sub-family specific 3' end and 10x G residues at the 5' end, the efficiency of the labelling reaction could be increased ten fold. Degenerate sub-family centred PCR was performed using the Kir 2.0 subclones as template, labelled as described in Materials and Methods, and digested with Hinf I. Low molecular weight fragment (

    Journal: BMC Genomics

    Article Title: Multigene family isoform profiling from blood cell lineages

    doi: 10.1186/1471-2164-3-22

    Figure Lengend Snippet: Increasing efficiency of detection of low MW restriction fragments. Using Klenow DNA polymerase and a probe with a sub-family specific 3' end and 10x G residues at the 5' end, the efficiency of the labelling reaction could be increased ten fold. Degenerate sub-family centred PCR was performed using the Kir 2.0 subclones as template, labelled as described in Materials and Methods, and digested with Hinf I. Low molecular weight fragment (

    Article Snippet: Reagents and materials Kit for DNA isolation from peripheral blood cells, T vector, RQ1 RNase-free DNase I, T4 DNA polymerase I, and DNA polymerase I large (Klenow) fragment were from Promega (Southampton, UK).

    Techniques: Polymerase Chain Reaction, Molecular Weight