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  • 94
    Thermo Fisher chargeswitch pcr clean up
    Chargeswitch Pcr Clean Up, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 69 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 497 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 257 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: 98/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL polymerase chain reaction pcr clean up kit
    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 Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Axygen axyprep mag polymerase chain reaction pcr clean up kit
    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).
    Axyprep Mag Polymerase Chain Reaction Pcr Clean Up Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Beckman Coulter pcr polymerase chain reaction clean up
    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).
    Pcr Polymerase Chain Reaction Clean Up, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs polymerase chain reaction pcr clean up kit
    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 Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen axyprep mag pcr clean up
    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).
    Axyprep Mag Pcr Clean Up, supplied by Axygen, used in various techniques. Bioz Stars score: 90/100, based on 641 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: 94/100, based on 1694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen 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 Axygen, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare 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 GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 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 Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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

    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