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  • 79
    Promega pcr clean up system promega
    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).
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    Promega wizard sv gel and pcr clean up system
    IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and <t>PCR</t> primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by <t>DNA</t> sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P
    Wizard Sv Gel And Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up systems
    IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and <t>PCR</t> primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by <t>DNA</t> sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P
    Pcr Clean Up Systems, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up kit
    ZNF224 binds to miR-663a promoter A. miR-663a promoter sequence analysis. Two regions containing 5′-CAGC-3′ sequence were found within - 500 bp. Arrow indicates primer binding sites for <t>PCR.</t> B and C. FLAG-ZNF224 was transfected to HEK293 cells, and ChIP assay using normal or FLAG IgG was carried out. PCR was performed using miR-663a promoter primers between -350 bp and -89 bp. In addition, ChIPed <t>DNA</t> was analyzed with qPCR. Data are from at least three independent experiments (n=3). D. FLAG-ZNF224 (0.5 1, and 2 μg) was transfected to MCF-7 cells in a dose-dependent manner for 24 h, and miR-663a transcript level was examined by qRT-PCR. miR-663a transcript level was normalized to U6 RNA. Data are from at least three independent experiments (n=3).
    Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up column
    Molecular analysis of Xanthomonas strains. Total genomic <t>DNA</t> of different Xanthomonas ) or integron gene cassette <t>PCR.</t> ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.
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    Promega pcr clean up system kit
    Molecular analysis of Xanthomonas strains. Total genomic <t>DNA</t> of different Xanthomonas ) or integron gene cassette <t>PCR.</t> ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.
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    Promega wizard pcr clean up system
    <t>PCR</t> and <t>DNA</t> sequencing
    Wizard Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizardsv pcr clean up system
    <t>PCR</t> and <t>DNA</t> sequencing
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    Promega pcr clean up mini columns
    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the <t>DNA</t> binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and <t>qRT-PCR</t> was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.
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    Promega wizard pcr clean up kit
    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
    Wizard Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizardtm pcr clean up kit
    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
    Wizardtm Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system purification kit
    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
    Pcr Clean Up System Purification Kit, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard pcr clean up system kit
    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
    Wizard Pcr Clean Up System Kit, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard magnesil pcr clean up system
    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
    Wizard Magnesil Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard sv 96 pcr clean up system
    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
    Wizard Sv 96 Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into <t>pGEM-T</t> after <t>PCR</t> amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.
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    <t>PCR</t> amplification of CpG islands. ( A ) 294 bp human hMLH1 promoter from colon cancer cell line RKO; ( B ) 605 bp human Cox-2 promoter from gastric cancer cell line MKN45. M1, puC18 <t>DNA/</t> Hea III marker; M2, 1 kb DNA ladder (Gibco no. 15615-016); lanes 1 and 4, untreated DNA; lanes 2 and 3, bisulfite-modified template.
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    <t>PCR-SSCP</t> and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.
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    <t>PCR-SSCP</t> and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.
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    <t>PCR-SSCP</t> and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.
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    <t>PCR-SSCP</t> and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.
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    <t>PCR-SSCP</t> and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.
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    <t>PCR</t> ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: <t>DNA</t> ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.
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    <t>PCR</t> ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: <t>DNA</t> ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.
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    <t>PCR</t> ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: <t>DNA</t> ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.
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    <t>PCR</t> ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: <t>DNA</t> ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.
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    <t>PCR</t> ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: <t>DNA</t> ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.
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    KDNA signatures of the 447-bp minicircle fragment of L. infantum ( = L. chagasi ) strains isolated from the canine reservoir. Five µ l of the <t>LSSP-PCR</t> reaction products, were loaded in each lane of a 6% polyacrylamide gel and silver stained. Lanes 2 to 11: genotype I (lanes 2–3: CB2 and CP3); genotype II (lane 4: CP5); genotype III (lanes 5–6: CB7 and CP2); genotype IV (lane 7: CP6); genotype V (lane 8: CB3); genotype VI (lane 9: IPT1); genotype VII (lane 10: CB10); genotype VIII (lane 11: CB4). Migration of the markers of the 1 Kb <t>DNA</t> ladder (Life Technologies, Inc., Gaithersburg, MD) is shown in lane 1, with the following molecular sizes (from the bottom up): 75, 134, 154, 201, 220, 298, 344, 396, 506, 517, 1018 and 1636 bp. Genotypes are indicated by roman numbers.
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    KDNA signatures of the 447-bp minicircle fragment of L. infantum ( = L. chagasi ) strains isolated from the canine reservoir. Five µ l of the <t>LSSP-PCR</t> reaction products, were loaded in each lane of a 6% polyacrylamide gel and silver stained. Lanes 2 to 11: genotype I (lanes 2–3: CB2 and CP3); genotype II (lane 4: CP5); genotype III (lanes 5–6: CB7 and CP2); genotype IV (lane 7: CP6); genotype V (lane 8: CB3); genotype VI (lane 9: IPT1); genotype VII (lane 10: CB10); genotype VIII (lane 11: CB4). Migration of the markers of the 1 Kb <t>DNA</t> ladder (Life Technologies, Inc., Gaithersburg, MD) is shown in lane 1, with the following molecular sizes (from the bottom up): 75, 134, 154, 201, 220, 298, 344, 396, 506, 517, 1018 and 1636 bp. Genotypes are indicated by roman numbers.
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    Image Search Results


    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

    IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and PCR primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by DNA sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P

    Journal: Nucleic Acids Research

    Article Title: IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner

    doi: 10.1093/nar/gky1012

    Figure Lengend Snippet: IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and PCR primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by DNA sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P

    Article Snippet: DNA was finally eluted using the WIZARD® SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR).

    Techniques: Expressing, Cross-linking Immunoprecipitation, CRISPR, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, DNA Sequencing, Quantitative RT-PCR, Negative Control, Western Blot, Modification, Purification, Transfection, Standard Deviation

    ZNF224 binds to miR-663a promoter A. miR-663a promoter sequence analysis. Two regions containing 5′-CAGC-3′ sequence were found within - 500 bp. Arrow indicates primer binding sites for PCR. B and C. FLAG-ZNF224 was transfected to HEK293 cells, and ChIP assay using normal or FLAG IgG was carried out. PCR was performed using miR-663a promoter primers between -350 bp and -89 bp. In addition, ChIPed DNA was analyzed with qPCR. Data are from at least three independent experiments (n=3). D. FLAG-ZNF224 (0.5 1, and 2 μg) was transfected to MCF-7 cells in a dose-dependent manner for 24 h, and miR-663a transcript level was examined by qRT-PCR. miR-663a transcript level was normalized to U6 RNA. Data are from at least three independent experiments (n=3).

    Journal: Oncotarget

    Article Title: ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a

    doi: 10.18632/oncotarget.8870

    Figure Lengend Snippet: ZNF224 binds to miR-663a promoter A. miR-663a promoter sequence analysis. Two regions containing 5′-CAGC-3′ sequence were found within - 500 bp. Arrow indicates primer binding sites for PCR. B and C. FLAG-ZNF224 was transfected to HEK293 cells, and ChIP assay using normal or FLAG IgG was carried out. PCR was performed using miR-663a promoter primers between -350 bp and -89 bp. In addition, ChIPed DNA was analyzed with qPCR. Data are from at least three independent experiments (n=3). D. FLAG-ZNF224 (0.5 1, and 2 μg) was transfected to MCF-7 cells in a dose-dependent manner for 24 h, and miR-663a transcript level was examined by qRT-PCR. miR-663a transcript level was normalized to U6 RNA. Data are from at least three independent experiments (n=3).

    Article Snippet: Finally, the DNA was purified with the PCR Clean-up Kit (Promega, WI, USA).

    Techniques: Sequencing, Binding Assay, Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Molecular analysis of Xanthomonas strains. Total genomic DNA of different Xanthomonas ) or integron gene cassette PCR. ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.

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

    Article Title: Integrons in Xanthomonas: A source of species genome diversity

    doi: 10.1073/pnas.0406620102

    Figure Lengend Snippet: Molecular analysis of Xanthomonas strains. Total genomic DNA of different Xanthomonas ) or integron gene cassette PCR. ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.

    Article Snippet: Amplified fragments were purified by using PCR clean-up columns (Promega), and DNA was sequenced directly by using the amplification primers or cloned into pGEM-T (Promega) according to the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight

    PCR and DNA sequencing

    Journal:

    Article Title: Production of a unique pneumococcal capsule serotype belonging to serogroup 6

    doi: 10.1099/mic.0.024521-0

    Figure Lengend Snippet: PCR and DNA sequencing

    Article Snippet: PCR products were purified using Wizard PCR Clean-up System (Promega), and the DNA sequencing was performed by the genomics core facility at the University of Alabama at Birmingham (UAB).

    Techniques: Polymerase Chain Reaction, DNA Sequencing

    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

    Journal: Cancer cell

    Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

    doi: 10.1016/j.ccr.2009.11.025

    Figure Lengend Snippet: Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

    Article Snippet: DNA samples were extracted using PCR clean-up mini-columns (Promega).

    Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Multiple Displacement Amplification

    Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

    Journal: Cancer cell

    Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

    doi: 10.1016/j.ccr.2009.11.025

    Figure Lengend Snippet: Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

    Article Snippet: DNA samples were extracted using PCR clean-up mini-columns (Promega).

    Techniques: Expressing, Transfection, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Amplification

    Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.

    Journal: PLoS ONE

    Article Title: Instability of the Octarepeat Region of the Human Prion Protein Gene

    doi: 10.1371/journal.pone.0026635

    Figure Lengend Snippet: Human PRNP octarepeat sequences and cloned octarepeats for instability analysis. (A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human PRNP octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human PRNP octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.

    Article Snippet: The PCR products were treated with the Wizard PCR clean-up kit (Promega, WI, USA), ligated to pGEM-T (for Pwo-amplified products, A-tailing was done first with Taq DNA polymerase in PCR buffer with 0.2 mM dATP) and transformed into competent DH5α cells (New England Biolabs, MA, USA) on LB-agar plates with X-gal.

    Techniques: Clone Assay, Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification

    Schematic diagrams for analysis of octarepeat mutation rate during PCR or replication in E.coli . (A) Measurement of octarepeat mutants resulting from PCR amplification of octarepeats. Cloned PrP ORF sequences (PrP-Oct5 and PrP-Oct11a) were subjected to PCR with primers HP20 and HP306r. The PCR products (depicted in a shaded box), which contained input (green line) and PCR-mutant (red line) octarepeat sequences as well as “paired” molecules (two parallel lines), were cleaned up and ligated to the pGEM-T vector, producing 4 kinds of ligation products: mutant monomer, wild type monomer, wild type dimer, and mutant dimer. The ligation products were transformed into DH5α competent cells, and the resulting colonies were directly examined by PCR with primers HP50f and HP293r. Plasmid DNAs were extracted from colonies containing mutant octarepeats, subjected to restriction analysis with Sac II and Spe I, and sequenced. The number of mutant colonies over the total number of colonies screened was calculated to represent the octarepeat mutation rate during the PCR process. Small oval: individual E.coli cell; big dashed-line oval: E.coli colony. (B) Measurement of octarepeat mutants resulting from DNA replication in E.coli . pOct5 or pOct11b was used to transform competent DH5α or XL-1 Red E.coli cells, and plasmid DNA sample prepared from a single colony (depicted in a shaded box) was used to transform competent DH5α cells. The mutant colonies (containing only mutant plasmid, red dashed-line oval) and new mutant colonies [containing some cells with the NEW replication-mutant octarepeat insert (light purple), light blue dashed-line oval] were screened out as in (A). The number of mutant colonies (excluding the new mutant colonies) over the total number of colonies examined should reflect the octarepeat mutation rate during the 1 st round of plasmid replication and cell division in E.coli (DH5α or XL-1 Red).

    Journal: PLoS ONE

    Article Title: Instability of the Octarepeat Region of the Human Prion Protein Gene

    doi: 10.1371/journal.pone.0026635

    Figure Lengend Snippet: Schematic diagrams for analysis of octarepeat mutation rate during PCR or replication in E.coli . (A) Measurement of octarepeat mutants resulting from PCR amplification of octarepeats. Cloned PrP ORF sequences (PrP-Oct5 and PrP-Oct11a) were subjected to PCR with primers HP20 and HP306r. The PCR products (depicted in a shaded box), which contained input (green line) and PCR-mutant (red line) octarepeat sequences as well as “paired” molecules (two parallel lines), were cleaned up and ligated to the pGEM-T vector, producing 4 kinds of ligation products: mutant monomer, wild type monomer, wild type dimer, and mutant dimer. The ligation products were transformed into DH5α competent cells, and the resulting colonies were directly examined by PCR with primers HP50f and HP293r. Plasmid DNAs were extracted from colonies containing mutant octarepeats, subjected to restriction analysis with Sac II and Spe I, and sequenced. The number of mutant colonies over the total number of colonies screened was calculated to represent the octarepeat mutation rate during the PCR process. Small oval: individual E.coli cell; big dashed-line oval: E.coli colony. (B) Measurement of octarepeat mutants resulting from DNA replication in E.coli . pOct5 or pOct11b was used to transform competent DH5α or XL-1 Red E.coli cells, and plasmid DNA sample prepared from a single colony (depicted in a shaded box) was used to transform competent DH5α cells. The mutant colonies (containing only mutant plasmid, red dashed-line oval) and new mutant colonies [containing some cells with the NEW replication-mutant octarepeat insert (light purple), light blue dashed-line oval] were screened out as in (A). The number of mutant colonies (excluding the new mutant colonies) over the total number of colonies examined should reflect the octarepeat mutation rate during the 1 st round of plasmid replication and cell division in E.coli (DH5α or XL-1 Red).

    Article Snippet: The PCR products were treated with the Wizard PCR clean-up kit (Promega, WI, USA), ligated to pGEM-T (for Pwo-amplified products, A-tailing was done first with Taq DNA polymerase in PCR buffer with 0.2 mM dATP) and transformed into competent DH5α cells (New England Biolabs, MA, USA) on LB-agar plates with X-gal.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Ligation, Transformation Assay

    PCR-amplified TCRBV2 transcripts from different sources were analyzed by SSCP. Lanes: 1, AB-TIL grown with IL-2 alone; 2, AB-TIL separately derived from a tissue fragment and grown with IL-2 and anti-CD3; 3, AB-TIL separately derived from a tissue fragment and grown with anti-CD3,8 bispecific mAbs and enriched for CD4 + cells; 4, freshly frozen tumor tissue from the shoulder metastatic lesion; 5, same as in lane 4 (duplicate conditions from a separate tumor tissue fragment); 6, AB-TIL separately derived from a tissue fragment and grown with anti-CD3,4 bispecific mAbs and highly enriched for CD8 + cells; 7, SH-TIL grown with anti-CD3,4 bispecific mAbs and highly enriched for CD8 + cells; 8, PBLs grown with anti-CD3,4 bispecific mAbs and highly enriched for CD8 + cells; 9, cytotoxic clone AB-1.

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

    Article Title: In vivo accumulation of the same anti-melanoma T cell clone in two different metastatic sites

    doi:

    Figure Lengend Snippet: PCR-amplified TCRBV2 transcripts from different sources were analyzed by SSCP. Lanes: 1, AB-TIL grown with IL-2 alone; 2, AB-TIL separately derived from a tissue fragment and grown with IL-2 and anti-CD3; 3, AB-TIL separately derived from a tissue fragment and grown with anti-CD3,8 bispecific mAbs and enriched for CD4 + cells; 4, freshly frozen tumor tissue from the shoulder metastatic lesion; 5, same as in lane 4 (duplicate conditions from a separate tumor tissue fragment); 6, AB-TIL separately derived from a tissue fragment and grown with anti-CD3,4 bispecific mAbs and highly enriched for CD8 + cells; 7, SH-TIL grown with anti-CD3,4 bispecific mAbs and highly enriched for CD8 + cells; 8, PBLs grown with anti-CD3,4 bispecific mAbs and highly enriched for CD8 + cells; 9, cytotoxic clone AB-1.

    Article Snippet: PCR products from V-region transcripts of TIL clones were purified from low-melting temperature agarose gels with the Wizard Prep PCR clean-up kit (Promega).

    Techniques: Polymerase Chain Reaction, Amplification, Derivative Assay

    PCR amplification of CpG islands. ( A ) 294 bp human hMLH1 promoter from colon cancer cell line RKO; ( B ) 605 bp human Cox-2 promoter from gastric cancer cell line MKN45. M1, puC18 DNA/ Hea III marker; M2, 1 kb DNA ladder (Gibco no. 15615-016); lanes 1 and 4, untreated DNA; lanes 2 and 3, bisulfite-modified template.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

    doi:

    Figure Lengend Snippet: PCR amplification of CpG islands. ( A ) 294 bp human hMLH1 promoter from colon cancer cell line RKO; ( B ) 605 bp human Cox-2 promoter from gastric cancer cell line MKN45. M1, puC18 DNA/ Hea III marker; M2, 1 kb DNA ladder (Gibco no. 15615-016); lanes 1 and 4, untreated DNA; lanes 2 and 3, bisulfite-modified template.

    Article Snippet: One microgram of genomic DNA was treated with sodium bisulfite as described ( ) and the Wizard DNA Clean-Up System Kit (A7280; Promega), subsequently used prior to PCR amplification.

    Techniques: Polymerase Chain Reaction, Amplification, Marker, Modification

    Detection of methylation in the hMLH1 promoter by Bst UI COBRA assay in the RKO and PACM82 cell lines. DNA without or with bisulfite treatment was amplified by PCR or ssPCR. The amplicon mixture was digested with Bst UI at 60°C for 3 h. M1, PCR and ssPCR as in Figure 1.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

    doi:

    Figure Lengend Snippet: Detection of methylation in the hMLH1 promoter by Bst UI COBRA assay in the RKO and PACM82 cell lines. DNA without or with bisulfite treatment was amplified by PCR or ssPCR. The amplicon mixture was digested with Bst UI at 60°C for 3 h. M1, PCR and ssPCR as in Figure 1.

    Article Snippet: One microgram of genomic DNA was treated with sodium bisulfite as described ( ) and the Wizard DNA Clean-Up System Kit (A7280; Promega), subsequently used prior to PCR amplification.

    Techniques: Methylation, Combined Bisulfite Restriction Analysis Assay, Amplification, Polymerase Chain Reaction

    PCR-SSCP and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.

    Journal: PLoS ONE

    Article Title: Identification and Analysis of Genetic Variations in Pri-MiRNAs Expressed Specifically or at a High Level in Sheep Skeletal Muscle

    doi: 10.1371/journal.pone.0117327

    Figure Lengend Snippet: PCR-SSCP and sequencing of pri-miR-133a (A), pri-miR-29a (B) and pri-miR-27b (C). The boxes in the sequencing trace show the genetic variations. CCC (+/−) in (A) and TAATAATAC (+/−) in (B) appear mixed, with disordered peaks due to the deletion locus. GC in (C) appears as double peaks because the genetic variations occurred in one chromosome, but not the other.

    Article Snippet: The PCR products that contained different genetic variations were chosen from the SSCP results, separated on a 1.5% agarose gel, purified with Agarose Gel PCR Fragment Clean-up system (Promega, Madison, WI, USA) and sequenced in both directions using Sanger sequencing (BGI, Beijing, China).

    Techniques: Polymerase Chain Reaction, Sequencing, Countercurrent Chromatography

    PCR ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: DNA ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.

    Journal: Journal of Clinical Laboratory Analysis

    Article Title: CTNND1 755 T > G Promoter Polymorphism and Risk of Pancreatic Carcinoma in Chinese

    doi: 10.1002/jcla.22055

    Figure Lengend Snippet: PCR ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: DNA ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.

    Article Snippet: The PCR fragments were recovered from agarose gel followed by purification with a DNA clean‐up kit (Wizard SV Gel and PCR Clean‐up System, Promega).

    Techniques: Polymerase Chain Reaction

    KDNA signatures of the 447-bp minicircle fragment of L. infantum ( = L. chagasi ) strains isolated from the canine reservoir. Five µ l of the LSSP-PCR reaction products, were loaded in each lane of a 6% polyacrylamide gel and silver stained. Lanes 2 to 11: genotype I (lanes 2–3: CB2 and CP3); genotype II (lane 4: CP5); genotype III (lanes 5–6: CB7 and CP2); genotype IV (lane 7: CP6); genotype V (lane 8: CB3); genotype VI (lane 9: IPT1); genotype VII (lane 10: CB10); genotype VIII (lane 11: CB4). Migration of the markers of the 1 Kb DNA ladder (Life Technologies, Inc., Gaithersburg, MD) is shown in lane 1, with the following molecular sizes (from the bottom up): 75, 134, 154, 201, 220, 298, 344, 396, 506, 517, 1018 and 1636 bp. Genotypes are indicated by roman numbers.

    Journal: PLoS ONE

    Article Title: KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

    doi: 10.1371/journal.pone.0043363

    Figure Lengend Snippet: KDNA signatures of the 447-bp minicircle fragment of L. infantum ( = L. chagasi ) strains isolated from the canine reservoir. Five µ l of the LSSP-PCR reaction products, were loaded in each lane of a 6% polyacrylamide gel and silver stained. Lanes 2 to 11: genotype I (lanes 2–3: CB2 and CP3); genotype II (lane 4: CP5); genotype III (lanes 5–6: CB7 and CP2); genotype IV (lane 7: CP6); genotype V (lane 8: CB3); genotype VI (lane 9: IPT1); genotype VII (lane 10: CB10); genotype VIII (lane 11: CB4). Migration of the markers of the 1 Kb DNA ladder (Life Technologies, Inc., Gaithersburg, MD) is shown in lane 1, with the following molecular sizes (from the bottom up): 75, 134, 154, 201, 220, 298, 344, 396, 506, 517, 1018 and 1636 bp. Genotypes are indicated by roman numbers.

    Article Snippet: LSSP-PCR Analysis For the production of kDNA signatures from the 447 bp L. infantum kDNA fragments, PCR products were purified by using the kit Wizard® PCR Preps – DNA purification System (Promega).

    Techniques: Isolation, Polymerase Chain Reaction, Staining, Migration

    KDNA signatures of the 447-bp minicircle fragment of L. infantum ( = L. chagasi ) strains isolated from human patients. Five µ l of the LSSP-PCR reaction products, were loaded in each lane of a 6% polyacrylamide gel and silver stained. Lanes 2 to 17: genotype I (lanes 2–4: HP7, HB5 and HB9); genotype II (lanes 5–6: HP10 and HP1); genotype III (lanes 7–8: HP6 and HB6); genotype VI (lane 9: HP5); genotype VII (lane 10: HB8); genotype VIII (lane 11: HP3); genotype IX (lane 12: HP8); genotype IV (lanes 13–14: HB4 and HB9); genotype V (lanes 15–16: HP4 and PP75); lane 17 the negative control of the LSSP-PCR reaction. Migration of the markers of the 1 Kb DNA ladder (Life Technologies, Inc., Gaithersburg, MD) is shown in lane 1, with the following molecular sizes (from the bottom up): 75, 134, 154, 201, 220, 298, 344, 396, 506, 517, 1018 and 1636 bp. Genotypes are indicated by roman numbers.

    Journal: PLoS ONE

    Article Title: KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

    doi: 10.1371/journal.pone.0043363

    Figure Lengend Snippet: KDNA signatures of the 447-bp minicircle fragment of L. infantum ( = L. chagasi ) strains isolated from human patients. Five µ l of the LSSP-PCR reaction products, were loaded in each lane of a 6% polyacrylamide gel and silver stained. Lanes 2 to 17: genotype I (lanes 2–4: HP7, HB5 and HB9); genotype II (lanes 5–6: HP10 and HP1); genotype III (lanes 7–8: HP6 and HB6); genotype VI (lane 9: HP5); genotype VII (lane 10: HB8); genotype VIII (lane 11: HP3); genotype IX (lane 12: HP8); genotype IV (lanes 13–14: HB4 and HB9); genotype V (lanes 15–16: HP4 and PP75); lane 17 the negative control of the LSSP-PCR reaction. Migration of the markers of the 1 Kb DNA ladder (Life Technologies, Inc., Gaithersburg, MD) is shown in lane 1, with the following molecular sizes (from the bottom up): 75, 134, 154, 201, 220, 298, 344, 396, 506, 517, 1018 and 1636 bp. Genotypes are indicated by roman numbers.

    Article Snippet: LSSP-PCR Analysis For the production of kDNA signatures from the 447 bp L. infantum kDNA fragments, PCR products were purified by using the kit Wizard® PCR Preps – DNA purification System (Promega).

    Techniques: Isolation, Polymerase Chain Reaction, Staining, Negative Control, Migration