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  • 99
    Thermo Fisher chargeswitch pcr clean up
    Chargeswitch Pcr Clean Up, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute polymerase chain reaction pcr clean up kit
    Enrichment of selected islet genes in LCM samples. mRNA expression genes was determined by quantitative TaqMan <t>RT-PCR</t> for each gene and for Actb from LCM islets (white bars) and total pancreas (black bars) of 8-week-old mice. Expression levels are presented as change in C t or the cycle number of each gene subtracted from the cycle number of beta-actin ( C t value 20.15) for the same <t>cDNA</t> sample. Lower numbers thus correspond to higher expression. Data are means ± SE from five CHF-fed NZL mice. * p ≤ 0.05
    Genelute Polymerase Chain Reaction Pcr Clean Up Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr clean up
    Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c <t>PCR</t> analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp <t>DNA</t> ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)
    Pcr Clean Up, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega polymerase chain reaction pcr clean up system
    The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter <t>nucleosome</t> repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by <t>qRT-PCR.</t> Expression levels were normalized to the maximum expression levels in wild-type (10 min).
    Polymerase Chain Reaction Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL polymerase chain reaction 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.
    Polymerase Chain Reaction Pcr Clean Up Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 95/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter pcr polymerase chain reaction clean up
    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 Polymerase Chain Reaction Clean Up, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen axyprep mag polymerase chain reaction 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.
    Axyprep Mag Polymerase Chain Reaction Pcr Clean Up Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 96/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen axyprep mag pcr clean up
    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.
    Axyprep Mag Pcr Clean Up, supplied by Axygen, used in various techniques. Bioz Stars score: 99/100, based on 515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
    Pcr Clean Up, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 99/100, based on 1430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelutetm pcr clean up kit
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
    Genelutetm Pcr Clean Up Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 205 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 762 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare pcr clean up
    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, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr clean up
    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, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen pcr clean up
    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, supplied by Axygen, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up
    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, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up columns
    Characterization of a stable <t>eys</t> rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of <t>RT-PCR</t> analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
    Pcr Clean Up Columns, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 95/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen pcr clean up kit
    Comparison of the sensitivities of mutation detection by <t>PCR</t> / RNP , PCR /T7 EI and direct Sanger sequencing. (a) The sg‐Os PDS ‐1 target site is located in exon1 of Os PDS . The PAM sequence is highlighted in red. ‘D1’ indicates a 1 bp deletion at the target site. (b c) Mixtures of WT and D1 PCR amplicons in different ratios were treated by PCR / RNP and PCR /T7 EI . (d) <t>DNA</t> sequence of the PCR amplicons surrounding the sg‐Os PDS ‐1 target site. The sg RNA sequence and 12 consecutive T's in the amplicons are highlighted in red and blue, respectively. (e) Mixtures of different proportions of WT and D1 PCR amplicons sequenced by the Sanger method.
    Pcr Clean Up Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 95/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GMbiolab pcr clean up kit
    Comparison of the sensitivities of mutation detection by <t>PCR</t> / RNP , PCR /T7 EI and direct Sanger sequencing. (a) The sg‐Os PDS ‐1 target site is located in exon1 of Os PDS . The PAM sequence is highlighted in red. ‘D1’ indicates a 1 bp deletion at the target site. (b c) Mixtures of WT and D1 PCR amplicons in different ratios were treated by PCR / RNP and PCR /T7 EI . (d) <t>DNA</t> sequence of the PCR amplicons surrounding the sg‐Os PDS ‐1 target site. The sg RNA sequence and 12 consecutive T's in the amplicons are highlighted in red and blue, respectively. (e) Mixtures of different proportions of WT and D1 PCR amplicons sequenced by the Sanger method.
    Pcr Clean Up Kit, supplied by GMbiolab, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up kit
    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> 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 <t>DNA</t> 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).
    Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific pcr clean up system
    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> 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 <t>DNA</t> 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).
    Pcr Clean Up System, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> 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 <t>DNA</t> 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).
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    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> 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 <t>DNA</t> 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).
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    Working scheme of Pre-S Gene Chip analysis . The virus <t>DNA</t> is extracted from the patient's blood or liver tissue. The pre-S region is <t>PCR-amplified</t> using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.
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    Working scheme of Pre-S Gene Chip analysis . The virus <t>DNA</t> is extracted from the patient's blood or liver tissue. The pre-S region is <t>PCR-amplified</t> using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.
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    Working scheme of Pre-S Gene Chip analysis . The virus <t>DNA</t> is extracted from the patient's blood or liver tissue. The pre-S region is <t>PCR-amplified</t> using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.
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    Working scheme of Pre-S Gene Chip analysis . The virus <t>DNA</t> is extracted from the patient's blood or liver tissue. The pre-S region is <t>PCR-amplified</t> using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.
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    Working scheme of Pre-S Gene Chip analysis . The virus <t>DNA</t> is extracted from the patient's blood or liver tissue. The pre-S region is <t>PCR-amplified</t> using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.
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    Working scheme of Pre-S Gene Chip analysis . The virus <t>DNA</t> is extracted from the patient's blood or liver tissue. The pre-S region is <t>PCR-amplified</t> using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.
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    Image Search Results


    Enrichment of selected islet genes in LCM samples. mRNA expression genes was determined by quantitative TaqMan RT-PCR for each gene and for Actb from LCM islets (white bars) and total pancreas (black bars) of 8-week-old mice. Expression levels are presented as change in C t or the cycle number of each gene subtracted from the cycle number of beta-actin ( C t value 20.15) for the same cDNA sample. Lower numbers thus correspond to higher expression. Data are means ± SE from five CHF-fed NZL mice. * p ≤ 0.05

    Journal: Diabetologia

    Article Title: Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome

    doi: 10.1007/s00125-009-1576-4

    Figure Lengend Snippet: Enrichment of selected islet genes in LCM samples. mRNA expression genes was determined by quantitative TaqMan RT-PCR for each gene and for Actb from LCM islets (white bars) and total pancreas (black bars) of 8-week-old mice. Expression levels are presented as change in C t or the cycle number of each gene subtracted from the cycle number of beta-actin ( C t value 20.15) for the same cDNA sample. Lower numbers thus correspond to higher expression. Data are means ± SE from five CHF-fed NZL mice. * p ≤ 0.05

    Article Snippet: The amplified cDNA was purified with a PCR clean-up kit (GenElute; Sigma-Aldrich).

    Techniques: Laser Capture Microdissection, Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    5′truncation profile of R2 elements in 0B and B+ (1–4) individuals from five populations of E. plorans . R2 element diagram shows the location of R2 PCR primers used to determine the length of 5'-truncated and full-length R2 elements. The collection of 5'-truncated and full-length R2 elements found in each individual has been represented in a summary diagram as in [59] . Each horizontal line represents the full-length R2 element in an individual, and the vertical lines indicate 5'-truncations. The asterisk indicates a 5′ truncation fixed or nearly fixed in all five populations.

    Journal: PLoS ONE

    Article Title: Preferential Occupancy of R2 Retroelements on the B Chromosomes of the Grasshopper Eyprepocnemis plorans

    doi: 10.1371/journal.pone.0091820

    Figure Lengend Snippet: 5′truncation profile of R2 elements in 0B and B+ (1–4) individuals from five populations of E. plorans . R2 element diagram shows the location of R2 PCR primers used to determine the length of 5'-truncated and full-length R2 elements. The collection of 5'-truncated and full-length R2 elements found in each individual has been represented in a summary diagram as in [59] . Each horizontal line represents the full-length R2 element in an individual, and the vertical lines indicate 5'-truncations. The asterisk indicates a 5′ truncation fixed or nearly fixed in all five populations. "microB" indicates 5' truncations observed in R2 copies contained in DNA microdissected from the B 24 chromosome in Torrox.

    Article Snippet: To confirm that the PCR amplification products corresponded to R2, the products were visualized in 1.5% agarose gel electrophoresis, cleaned with Gen Elute PCR Clean-Up Kit (Sigma), cloned into the TOPO TA vector (Invitrogen), and sequenced (Macrogen).

    Techniques: Polymerase Chain Reaction

    PCR analysis for the presence of R2 transcripts. R2 elements were observed in the cDNA obtained from ovaries (ov) and eggs (eg), but not in female somatic body (fb), embryos (em) and six male tissues (not shown). Ø = Negative control (with no cDNA), C+ = Positive control (genomic DNA).

    Journal: PLoS ONE

    Article Title: Preferential Occupancy of R2 Retroelements on the B Chromosomes of the Grasshopper Eyprepocnemis plorans

    doi: 10.1371/journal.pone.0091820

    Figure Lengend Snippet: PCR analysis for the presence of R2 transcripts. R2 elements were observed in the cDNA obtained from ovaries (ov) and eggs (eg), but not in female somatic body (fb), embryos (em) and six male tissues (not shown). Ø = Negative control (with no cDNA), C+ = Positive control (genomic DNA).

    Article Snippet: To confirm that the PCR amplification products corresponded to R2, the products were visualized in 1.5% agarose gel electrophoresis, cleaned with Gen Elute PCR Clean-Up Kit (Sigma), cloned into the TOPO TA vector (Invitrogen), and sequenced (Macrogen).

    Techniques: Polymerase Chain Reaction, Negative Control, Positive Control

    Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c PCR analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp DNA ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c PCR analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp DNA ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Generated, Electroporation, CRISPR, Knock-Out, Polymerase Chain Reaction, Non-Homologous End Joining, Sequencing, Marker, Transmission Assay

    One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d Time-schedule comparisons of targeting methods using ES cells by CRISPR in B6 oocytes, and CLICK in Cre oocytes. CLICK saves about 6 months of crossing and reduces breeding costs during the study period (Additional file 13 : Figure S13)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d Time-schedule comparisons of targeting methods using ES cells by CRISPR in B6 oocytes, and CLICK in Cre oocytes. CLICK saves about 6 months of crossing and reduces breeding costs during the study period (Additional file 13 : Figure S13)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Knock-Out, In Vitro, Mouse Assay, Polymerase Chain Reaction, Electroporation, Sequencing, Expressing, Marker, CRISPR

    Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2 : Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15 : Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3 : Figure S3)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2 : Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15 : Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3 : Figure S3)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Generated, CRISPR, Polymerase Chain Reaction, Sequencing, Transmission Assay, Marker

    The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter nucleosome repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by qRT-PCR. Expression levels were normalized to the maximum expression levels in wild-type (10 min).

    Journal: Nucleic Acids Research

    Article Title: INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes

    doi: 10.1093/nar/gkw1292

    Figure Lengend Snippet: The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter nucleosome repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by qRT-PCR. Expression levels were normalized to the maximum expression levels in wild-type (10 min).

    Article Snippet: Mono nucleosome DNA bands were purified (Wizard® SV Gel and polymerase chain reaction (PCR) Clean-Up System, Promega).

    Techniques: Variant Assay, Mutagenesis, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

    H 2 O 2 production and release by cultured KGN cells and effects of exogenous H 2 O 2 . ( A ) NOX4 RT-PCR analysis and Western blot of cultured KGN cells show single bands of 160 bp and 68 kDa, respectively. Controls using RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Hydrogen peroxide assay of untreated KGN cells showed increasing H 2 O 2 levels in the supernatant over a time period of 2 h ( n = 6). Signal intensities were normalized to start point values. Bars indicate SEM. ( C ) Exogenously added H 2 O 2 reduced cell viability in a dose dependent manner. Cell counts after treatment of KGN cells with different concentrations of H 2 O 2 for 24 h ( n = 2–5 for each concentration) are shown with an interpolated sigmoidal standard curve ( r 2 = 0.9361). Bars indicate SEM. ( D ) Images of KGN cells treated with H 2 O 2 (1 mM) for 24 h compared to untreated control cells. Scale bars indicate 200 µm.

    Journal: Antioxidants

    Article Title: A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

    doi: 10.3390/antiox8110518

    Figure Lengend Snippet: H 2 O 2 production and release by cultured KGN cells and effects of exogenous H 2 O 2 . ( A ) NOX4 RT-PCR analysis and Western blot of cultured KGN cells show single bands of 160 bp and 68 kDa, respectively. Controls using RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Hydrogen peroxide assay of untreated KGN cells showed increasing H 2 O 2 levels in the supernatant over a time period of 2 h ( n = 6). Signal intensities were normalized to start point values. Bars indicate SEM. ( C ) Exogenously added H 2 O 2 reduced cell viability in a dose dependent manner. Cell counts after treatment of KGN cells with different concentrations of H 2 O 2 for 24 h ( n = 2–5 for each concentration) are shown with an interpolated sigmoidal standard curve ( r 2 = 0.9361). Bars indicate SEM. ( D ) Images of KGN cells treated with H 2 O 2 (1 mM) for 24 h compared to untreated control cells. Scale bars indicate 200 µm.

    Article Snippet: Amplicon identity was verified by agarose gel electrophoresis, consecutive cDNA extraction with Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) and sequence analysis (GATC, Konstanz, Germany).

    Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, H2O2 Assay, Concentration Assay

    KGN cells express functional TRPM2. ( A ) TRPM2 RT-PCR shows a band at 103 bp. Controls with RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Addition of H 2 O 2 (1 mM) increased the fluorescence signal of the four individual KGN cells shown, which were loaded with the Ca 2+ -sensitive dye Fluoforte. Background signals were subtracted and fluorescence is shown relative to the respective start value of each region of interest (ROI). ( C ) Fluorescence images, taken before ( a ) and after ( b ) the first stimulation with H 2 O 2 . ( D ) Treatment with the inhibitor (ACA; 20 µM), 4 h prior to and during the measurement, blocked the Ca 2+ increase upon stimulation with H 2 O 2 , but not with 0.05‰ trypsin (T). Images ( c – f ) represent the indicated time points. ( E ) The H 2 O 2 -derived Ca 2+ increase was obtained in the DMSO control and thus ruled out solvent effects. Images ( g – j ) represent the indicated time points. The pseudo-color scale shown in ( c ) applies for all live cell images. Colored frames mark the cells represented in the corresponding graphs. Scale bars indicate 50 µm.

    Journal: Antioxidants

    Article Title: A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

    doi: 10.3390/antiox8110518

    Figure Lengend Snippet: KGN cells express functional TRPM2. ( A ) TRPM2 RT-PCR shows a band at 103 bp. Controls with RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Addition of H 2 O 2 (1 mM) increased the fluorescence signal of the four individual KGN cells shown, which were loaded with the Ca 2+ -sensitive dye Fluoforte. Background signals were subtracted and fluorescence is shown relative to the respective start value of each region of interest (ROI). ( C ) Fluorescence images, taken before ( a ) and after ( b ) the first stimulation with H 2 O 2 . ( D ) Treatment with the inhibitor (ACA; 20 µM), 4 h prior to and during the measurement, blocked the Ca 2+ increase upon stimulation with H 2 O 2 , but not with 0.05‰ trypsin (T). Images ( c – f ) represent the indicated time points. ( E ) The H 2 O 2 -derived Ca 2+ increase was obtained in the DMSO control and thus ruled out solvent effects. Images ( g – j ) represent the indicated time points. The pseudo-color scale shown in ( c ) applies for all live cell images. Colored frames mark the cells represented in the corresponding graphs. Scale bars indicate 50 µm.

    Article Snippet: Amplicon identity was verified by agarose gel electrophoresis, consecutive cDNA extraction with Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) and sequence analysis (GATC, Konstanz, Germany).

    Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Derivative Assay

    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

    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

    Recovery of three distinct coat-colour mutations by CRISPR/Cas. ( a ) Schematic illustration of three coat-colour mutations in rats. albino ( Tyr c ): SNP missense mutation in the Tyr gene. non-agouti ( Asip a ): 19-bp deletion in exon 2 of the Agouti signalling protein ( Asip ) gene. hooded ( Kit h ): integration of an 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. ( b ) Coat-colour phenotypes ( C, a, h ) recovered from albino by injecting gRNA: Tyr c , Cas9 mRNA, and ssODN of the Tyr C allele into F344 rat embryos ( c, a, h ). ( c ) Sequence analysis of the targeted Tyr exon 2 in the injected F344 rats. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise HDR-mediated SNP exchange of Tyr C . ( d ) Recovery of the non-agouti phenotype by injecting gRNA: Asip a , Cas9 mRNA, and ssODN of the Asip A allele into F344 rat embryos. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise short DNA fragment integration of the Asip A gene . ( e ) Recovery of the hooded mutation by injecting gRNA: Kit h -1 , gRNA: Kit h -2 , and ssODN of the Kit H allele into F344 rat embryos. Cas9 and two gRNAs with ssODN mediated the introduction of indel mutations at the targeted Kit h -1 locus, and the precise large deletion between the two cutting edges of Kit H . ( f ) PCR analysis of the injected F344 rats using primers designed against each outer side of the two LTR sequences. M: DNA marker phiX174-HaeIII digest.

    Journal: Nature Communications

    Article Title: Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform

    doi: 10.1038/ncomms5240

    Figure Lengend Snippet: Recovery of three distinct coat-colour mutations by CRISPR/Cas. ( a ) Schematic illustration of three coat-colour mutations in rats. albino ( Tyr c ): SNP missense mutation in the Tyr gene. non-agouti ( Asip a ): 19-bp deletion in exon 2 of the Agouti signalling protein ( Asip ) gene. hooded ( Kit h ): integration of an 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. ( b ) Coat-colour phenotypes ( C, a, h ) recovered from albino by injecting gRNA: Tyr c , Cas9 mRNA, and ssODN of the Tyr C allele into F344 rat embryos ( c, a, h ). ( c ) Sequence analysis of the targeted Tyr exon 2 in the injected F344 rats. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise HDR-mediated SNP exchange of Tyr C . ( d ) Recovery of the non-agouti phenotype by injecting gRNA: Asip a , Cas9 mRNA, and ssODN of the Asip A allele into F344 rat embryos. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise short DNA fragment integration of the Asip A gene . ( e ) Recovery of the hooded mutation by injecting gRNA: Kit h -1 , gRNA: Kit h -2 , and ssODN of the Kit H allele into F344 rat embryos. Cas9 and two gRNAs with ssODN mediated the introduction of indel mutations at the targeted Kit h -1 locus, and the precise large deletion between the two cutting edges of Kit H . ( f ) PCR analysis of the injected F344 rats using primers designed against each outer side of the two LTR sequences. M: DNA marker phiX174-HaeIII digest.

    Article Snippet: To prepare Cas9 mRNA and gRNA, T7-promoter Cas9 and gRNA expression plasmids were linearized with Xho I and Hind III, respectively, and extracted with NucleoSpin Gel and PCR Clean-up kits (Macherey-Nagel, Düren, Germany).

    Techniques: CRISPR, Mutagenesis, Sequencing, Injection, Polymerase Chain Reaction, Marker

    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Journal: Scientific Reports

    Article Title: In vivo targeted single-nucleotide editing in zebrafish

    doi: 10.1038/s41598-018-29794-9

    Figure Lengend Snippet: Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Article Snippet: The PCR products from the chd and oep amplifications were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced by Fasmac (Atsugi, Japan) or by Macrogen Japan (Kyoto, Japan) with the chd _1st_F and oep _1st_F primers, respectively.

    Techniques: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing

    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

    Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Journal: PLoS ONE

    Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

    doi: 10.1371/journal.pone.0200789

    Figure Lengend Snippet: Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Article Snippet: To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

    Comparison of the sensitivities of mutation detection by PCR / RNP , PCR /T7 EI and direct Sanger sequencing. (a) The sg‐Os PDS ‐1 target site is located in exon1 of Os PDS . The PAM sequence is highlighted in red. ‘D1’ indicates a 1 bp deletion at the target site. (b c) Mixtures of WT and D1 PCR amplicons in different ratios were treated by PCR / RNP and PCR /T7 EI . (d) DNA sequence of the PCR amplicons surrounding the sg‐Os PDS ‐1 target site. The sg RNA sequence and 12 consecutive T's in the amplicons are highlighted in red and blue, respectively. (e) Mixtures of different proportions of WT and D1 PCR amplicons sequenced by the Sanger method.

    Journal: Plant Biotechnology Journal

    Article Title: Genotyping genome‐edited mutations in plants using CRISPR ribonucleoprotein complexes

    doi: 10.1111/pbi.12938

    Figure Lengend Snippet: Comparison of the sensitivities of mutation detection by PCR / RNP , PCR /T7 EI and direct Sanger sequencing. (a) The sg‐Os PDS ‐1 target site is located in exon1 of Os PDS . The PAM sequence is highlighted in red. ‘D1’ indicates a 1 bp deletion at the target site. (b c) Mixtures of WT and D1 PCR amplicons in different ratios were treated by PCR / RNP and PCR /T7 EI . (d) DNA sequence of the PCR amplicons surrounding the sg‐Os PDS ‐1 target site. The sg RNA sequence and 12 consecutive T's in the amplicons are highlighted in red and blue, respectively. (e) Mixtures of different proportions of WT and D1 PCR amplicons sequenced by the Sanger method.

    Article Snippet: Production of sgRNA and crRNA Templates for transcription of sgRNA and crRNA were amplified using corresponding primer sets (Table ) with high‐fidelity DNA polymerase and purified with PCR Clean‐Up Kit (Axygen). sgRNA and crRNA were synthesized using the HiScribe T7 In Vitro Transcription Kit (New England Biolabs) according to the manufacturer's instructions and purified by ethanol precipitation method.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Sequencing

    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

    Working scheme of Pre-S Gene Chip analysis . The virus DNA is extracted from the patient's blood or liver tissue. The pre-S region is PCR-amplified using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.

    Journal: Journal of Biomedical Science

    Article Title: A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers

    doi: 10.1186/1423-0127-16-84

    Figure Lengend Snippet: Working scheme of Pre-S Gene Chip analysis . The virus DNA is extracted from the patient's blood or liver tissue. The pre-S region is PCR-amplified using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.

    Article Snippet: For the DNA sequencing analysis, the colony PCR products were purified using a kit (PCR Clean-Up Kit; Roche Applied Sciences, Indianapolis, IN) and sent to our university's DNA Sequencing Facility for analysis.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Labeling, Agarose Gel Electrophoresis, Staining, Nested PCR, Hybridization, TA Cloning, Plasmid Preparation, Clone Assay