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  • 99
    New England Biolabs monarch pcr dna cleanup kit
    Pooled measurement of <t>DNA</t> polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After <t>PCR</t> amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Monarch Pcr Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 650 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher chargeswitch pcr clean up kit
    Pooled measurement of <t>DNA</t> polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After <t>PCR</t> amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Chargeswitch Pcr Clean Up Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up kit
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
    Pcr Clean Up Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 6126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute pcr clean up kit
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
    Genelute Pcr Clean Up Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen pcr cleanup kit
    Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative <t>RT-PCR</t> analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic <t>DNA</t> samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.
    Pcr Cleanup Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen ultraclean pcr clean up kit
    Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative <t>RT-PCR</t> analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic <t>DNA</t> samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.
    Ultraclean Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1739 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system kit
    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the <t>PCR</t> products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.
    Pcr Clean Up System Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen pcr clean up kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Pcr Clean Up Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 830 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Axygen axyprep mag pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Axyprep Mag Pcr Clean Up Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 89/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen pcr cleanup kit
    Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the <t>T-DNA</t> insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and <t>RT-PCR</t> was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p
    Pcr Cleanup Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 94/100, based on 711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Axygen axyprep pcr cleanup kit
    Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the <t>T-DNA</t> insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and <t>RT-PCR</t> was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p
    Axyprep Pcr Cleanup Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 89/100, based on 394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Journal: Genome Biology

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats

    doi: 10.1186/s13059-020-02124-x

    Figure Lengend Snippet: Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Article Snippet: The PCR products from all reactions were purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions.

    Techniques: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

    Techniques: Purification, Chromatin Immunoprecipitation, Concentration Assay, Sensitive Assay, Polymerase Chain Reaction

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Sequencing, Polymerase Chain Reaction

    Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Journal: Viruses

    Article Title: A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

    doi: 10.3390/v11121129

    Figure Lengend Snippet: Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Article Snippet: Amplified DNA Clean Up REPLI-g samples were purified using the NucleoSpin Gel and PCR clean-up Kit (Macherey-Nagel Düren, Germany).

    Techniques: Polymerase Chain Reaction, Whole Genome Amplification

    Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 ( attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 ( attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).

    Journal: Applied and Environmental Microbiology

    Article Title: Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

    doi: 10.1128/AEM.00485-19

    Figure Lengend Snippet: Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 ( attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 ( attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).

    Article Snippet: DNA fragments were purified from agarose gels using the NucleoSpin gel and PCR cleanup kit from Macherey-Nagel.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight

    Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative RT-PCR analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic DNA samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.

    Journal: Genes & Development

    Article Title: Chromatin restriction by the nucleosome remodeler Mi-2β and functional interplay with lineage-specific transcription regulators control B-cell differentiation

    doi: 10.1101/gad.321901.118

    Figure Lengend Snippet: Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative RT-PCR analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic DNA samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.

    Article Snippet: Amplified DNA was purified with a Qiagen PCR cleanup kit.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Expressing, Polymerase Chain Reaction, Amplification, Southern Blot, Flow Cytometry, Cytometry, Cell Differentiation

    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the PCR products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Distinct Leishmania Species Infecting Wild Caviomorph Rodents (Rodentia: Hystricognathi) from Brazil

    doi: 10.1371/journal.pntd.0003389

    Figure Lengend Snippet: Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the PCR products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.

    Article Snippet: The PCR products obtained for HSP70 (234) targeting and the products positive only in kDNA targeting were purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega).

    Techniques: Amplification, Polymerase Chain Reaction, Electrophoresis, Staining, Molecular Weight, Marker, Infection, Negative Control, Mass Spectrometry

    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Journal: BioMed Research International

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico

    doi: 10.1155/2017/5170680

    Figure Lengend Snippet: Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Article Snippet: PCR amplified DNA was cleaned using PCR clean-up kit following the kit protocol (Promega, Wisconsin, USA), sequenced, and verified using nucleotide BLAST tool.

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction

    PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.

    Journal: Journal of Bacteriology

    Article Title: Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿

    doi: 10.1128/JB.00543-07

    Figure Lengend Snippet: PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.

    Article Snippet: Annealing was achieved by incubating 800 pmol of each oligonucleotide in T4 ligation buffer at 95°C for 3 min and allowing the reaction mixture to cool at room temperature for 5 h, after which the 3′ PA3719 DNA was purified using a gel and PCR cleanup kit (Promega).

    Techniques: In Vitro, Polymerase Chain Reaction, Generated, Purification, Electrophoresis, Produced

    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Journal: Epigenetics & Chromatin

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination

    doi: 10.1186/s13072-018-0247-4

    Figure Lengend Snippet: TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Article Snippet: Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick).

    Techniques: Cell Culture, Purification, Immunoprecipitation, Polymerase Chain Reaction

    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Journal: Nature Communications

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    doi: 10.1038/s41467-019-13193-3

    Figure Lengend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Article Snippet: After linearization by digestion with DraI, the DNA fragment was purified with PCR Clean-up Kit (Clontech Laboratories) and transcribed using AmpliScribe T7-Flash Transcription Kit (Lucigen).

    Techniques: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test

    Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the T-DNA insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and RT-PCR was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p

    Journal: Frontiers in Plant Science

    Article Title: Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis

    doi: 10.3389/fpls.2017.01813

    Figure Lengend Snippet: Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the T-DNA insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and RT-PCR was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p

    Article Snippet: After washing, the DNA-protein cross-links obtained were reversed at 65°C for 12 h, and DNA was purified using PCR Cleanup Kit (Axygen) for PCR reactions.

    Techniques: Over Expression, Expressing, Transgenic Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Microscopy, Software