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  • 90
    Thermo Fisher pcr clean up kit
    Pcr Clean Up Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega polymerase chain reaction pcr clean up system
    The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter <t>nucleosome</t> repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by <t>qRT-PCR.</t> Expression levels were normalized to the maximum expression levels in wild-type (10 min).
    Polymerase Chain Reaction Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system kit promega
    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the <t>PCR</t> products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.
    Pcr Clean Up System Kit Promega, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 21438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pcr clean up kit
    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic <t>DNA</t> level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).
    Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    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.
    Pcr Clean Up Column, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 129 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: 99/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    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.
    Pcr Clean Up Mini Columns, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega wizardtm pcr clean up kit
    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.
    Wizardtm Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizardsv pcr clean up system
    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.
    Wizardsv Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega wizard pcr clean up system kit
    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.
    Wizard Pcr Clean Up System Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system purification kit
    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.
    Pcr Clean Up System Purification Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega pcr clean up spin 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.
    Pcr Clean Up Spin Columns, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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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    Expression profiles of HwHog-ChIP positive genes in adapted and stressed H. werneckii cells and ChIP results in adapted cells . <t>RT-PCR</t> was performed with RNA isolated from adapted cells (Adaptation) or cells exposed to hypersaline stress (Hyperosmotic stress) as indicated. 26S rRNA ( Hw26SRR ) was used as an internal control for template normalization. The HwHog1-ChIP and sequential RNA polymerase II/HwHog1-ChIP (SeqChIP) results for cells adapted to 3 M NaCl and 4.5 M NaCl are presented in parallel. The negative control for nonspecific binding of antibodies in both ChIP experiments is represented in the DNA amplification from 100-fold diluted input samples is labeled as "INPUT". HwCOB1 and 26SRR genes were negative controls and HwGPD1A was the positive control for HwHog1 and RNA polymerase II cross-linking. " width="250" height="auto" />
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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> 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</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</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</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</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</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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    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

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

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0003389

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

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

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

    PCR verification of mt minichromosomes of Haematopinus suis and H. apri . ( A ) Lanes 1 and 14: low mass ladder (LML). Lanes 2 and 13: molecular weight marker VII (MWM). Lanes 3–10: amplicons from eight minichromosomes of H. suis (B2311), rrnS -trnC , trnL 1 - rrnL , nad2-trnI- cox1 -trnL 2 , trnR- nad4L -nad6-trnM , trnK-nad4-atp8 - atp6 -trnN , trnE - cob- trnV , trnQ - nad1 -trnT-trnG-nad3-trnW , and trnD-trnY-cox2-trnS1-trnS2-trnP- cox3 -trnA . Lanes 11–12: PCR amplicons from two minichromosomes of H. apri (B2418), rrnS -trnC , and trnQ - nad1 -trnT-trnG-nad3-trnW . Genes where PCR primers were designed are in bold. ( B ) Lane 1: amplicon from trnH- nad5 -trnF minichromosome of H. suis (B2311). Lanes 2, 9, and 15: 1-kb ladder. Lanes 3–8 and 10–14: PCR amplicons from seven minichromosomes of H. apri (B2418), trnK-nad4-atp8 - atp6 -trnN , trnK- nad4 -atp8-atp6-trnN , nad2-trnI- cox1 -trnL 2 , nad2 -trnI-cox1-trnL 2 , trnD-trnY- cox2 -trnS1-trnS2-trnP-cox3-trnA , trnD-trnY-cox2-trnS1-trnS2-trnP- cox3 -trnA , trnE- cob- trnV , trnH- nad5 -trnF , trnR- nad4L -nad6-trnM , trnR-nad4L- nad6 -trnM , and trnL 1 - rrnL . ( C ) Amplicons by pig-lice-specific primers, PGC1F-PGC1R and PG12SF-PG12SR, from the entire nad2-trnI-cox1-trnL 2 minichromosome (4.5 kb, Lane 1) and the entire rrnS-trnC minichromosome (3.5 kb, Lane 4) of H. suis . Lane 2: LML. Lane 3: 1-kb Ladder. ( D ) Amplicons from the coding regions of mt minichromosomes of H. suis and H. apri . Lanes 1 and 11: LML. Lanes 2 and 10: MWM. Lanes 3–4: H. suis from Australia (B2311). Lanes 5–6: H. suis from Poland (B2419). Lanes 7–8: H. apri from Japan (B4218). Lane 9: H. suis from China (B2572).

    Journal: Genome Biology and Evolution

    Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals

    doi: 10.1093/gbe/evt094

    Figure Lengend Snippet: PCR verification of mt minichromosomes of Haematopinus suis and H. apri . ( A ) Lanes 1 and 14: low mass ladder (LML). Lanes 2 and 13: molecular weight marker VII (MWM). Lanes 3–10: amplicons from eight minichromosomes of H. suis (B2311), rrnS -trnC , trnL 1 - rrnL , nad2-trnI- cox1 -trnL 2 , trnR- nad4L -nad6-trnM , trnK-nad4-atp8 - atp6 -trnN , trnE - cob- trnV , trnQ - nad1 -trnT-trnG-nad3-trnW , and trnD-trnY-cox2-trnS1-trnS2-trnP- cox3 -trnA . Lanes 11–12: PCR amplicons from two minichromosomes of H. apri (B2418), rrnS -trnC , and trnQ - nad1 -trnT-trnG-nad3-trnW . Genes where PCR primers were designed are in bold. ( B ) Lane 1: amplicon from trnH- nad5 -trnF minichromosome of H. suis (B2311). Lanes 2, 9, and 15: 1-kb ladder. Lanes 3–8 and 10–14: PCR amplicons from seven minichromosomes of H. apri (B2418), trnK-nad4-atp8 - atp6 -trnN , trnK- nad4 -atp8-atp6-trnN , nad2-trnI- cox1 -trnL 2 , nad2 -trnI-cox1-trnL 2 , trnD-trnY- cox2 -trnS1-trnS2-trnP-cox3-trnA , trnD-trnY-cox2-trnS1-trnS2-trnP- cox3 -trnA , trnE- cob- trnV , trnH- nad5 -trnF , trnR- nad4L -nad6-trnM , trnR-nad4L- nad6 -trnM , and trnL 1 - rrnL . ( C ) Amplicons by pig-lice-specific primers, PGC1F-PGC1R and PG12SF-PG12SR, from the entire nad2-trnI-cox1-trnL 2 minichromosome (4.5 kb, Lane 1) and the entire rrnS-trnC minichromosome (3.5 kb, Lane 4) of H. suis . Lane 2: LML. Lane 3: 1-kb Ladder. ( D ) Amplicons from the coding regions of mt minichromosomes of H. suis and H. apri . Lanes 1 and 11: LML. Lanes 2 and 10: MWM. Lanes 3–4: H. suis from Australia (B2311). Lanes 5–6: H. suis from Poland (B2419). Lanes 7–8: H. apri from Japan (B4218). Lane 9: H. suis from China (B2572).

    Article Snippet: PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega).

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Amplification

    Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).

    Journal: Journal of Experimental Botany

    Article Title: Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

    doi: 10.1093/jxb/err188

    Figure Lengend Snippet: Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).

    Article Snippet: After PCR purification using Wizard SV PCR Clean-up System (Promega), the amiRNA precursor (254 bp) was cloned into pGEM-T Easy (Promega) using E. coli DH5α.

    Techniques: Construct, Synthesized, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Molecular Weight, Sequencing

    Identification of osKaR insertion site Arbitrary-primed PCR (AP-PCR) is a quick method to precisely identify the genomic region where a transposon has inserted. The following is specific for osKaR insertions: genomic DNA of the osKaR mutant is extracted (see Basic Protocol 1) and used for a semi-random PCR using the osKaR -specific primer oPCR1 and the random primer Deg3. The Deg3 primer consists of an 11-nucleotide random primer (in blue) with a 25-nucleotide specific tail (in red). The resulting PCR product is purified and used for a 2 nd PCR using the osKaR -specific primer Anchor1 and the Deg3-tail specific primer Deg4. The resulting PCR product is purified and DNA sequencing is performed using the osKaR -specific primer Anchor2.

    Journal: Current protocols in microbiology

    Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)

    doi: 10.1002/9780471729259.mc09d03s30

    Figure Lengend Snippet: Identification of osKaR insertion site Arbitrary-primed PCR (AP-PCR) is a quick method to precisely identify the genomic region where a transposon has inserted. The following is specific for osKaR insertions: genomic DNA of the osKaR mutant is extracted (see Basic Protocol 1) and used for a semi-random PCR using the osKaR -specific primer oPCR1 and the random primer Deg3. The Deg3 primer consists of an 11-nucleotide random primer (in blue) with a 25-nucleotide specific tail (in red). The resulting PCR product is purified and used for a 2 nd PCR using the osKaR -specific primer Anchor1 and the Deg3-tail specific primer Deg4. The resulting PCR product is purified and DNA sequencing is performed using the osKaR -specific primer Anchor2.

    Article Snippet: Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282)

    Techniques: Polymerase Chain Reaction, Mutagenesis, Purification, DNA Sequencing

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

    Journal: BioMed Research International

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

    doi: 10.1155/2017/5170680

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

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

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction

    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

    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 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

    Expression profiles of HwHog-ChIP positive genes in adapted and stressed H. werneckii cells and ChIP results in adapted cells . RT-PCR was performed with RNA isolated from adapted cells (Adaptation) or cells exposed to hypersaline stress (Hyperosmotic stress) as indicated. 26S rRNA ( Hw26SRR ) was used as an internal control for template normalization. The HwHog1-ChIP and sequential RNA polymerase II/HwHog1-ChIP (SeqChIP) results for cells adapted to 3 M NaCl and 4.5 M NaCl are presented in parallel. The negative control for nonspecific binding of antibodies in both ChIP experiments is represented in the

    Journal: BMC Genomics

    Article Title: Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii

    doi: 10.1186/1471-2164-8-280

    Figure Lengend Snippet: Expression profiles of HwHog-ChIP positive genes in adapted and stressed H. werneckii cells and ChIP results in adapted cells . RT-PCR was performed with RNA isolated from adapted cells (Adaptation) or cells exposed to hypersaline stress (Hyperosmotic stress) as indicated. 26S rRNA ( Hw26SRR ) was used as an internal control for template normalization. The HwHog1-ChIP and sequential RNA polymerase II/HwHog1-ChIP (SeqChIP) results for cells adapted to 3 M NaCl and 4.5 M NaCl are presented in parallel. The negative control for nonspecific binding of antibodies in both ChIP experiments is represented in the "NoAB" line and the positive control for genomic DNA amplification from 100-fold diluted input samples is labeled as "INPUT". HwCOB1 and 26SRR genes were negative controls and HwGPD1A was the positive control for HwHog1 and RNA polymerase II cross-linking.

    Article Snippet: ChIP eluates and inputs were reverse cross-linked in 0.2 M NaCl for 5 h at 65°C, incubated with 20 μg of RNase for 30 min at 37°C, followed by treatment with 10 μg of proteinase K. DNA was purified using the Wizard PCR Clean-up Purification System (Promega) and eluted with 150 μL of water for Hog-ChIP samples or with 50 μL for SeqChIP samples.

    Techniques: Expressing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Isolation, Negative Control, Binding Assay, Positive Control, Amplification, Labeling

    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 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

    Representative methylation-specific polymerase chain reaction (MSP) results from primary ovarian carcinomas . PCR products in lane U indicate the presence of unmethylated alleles whereas PCR products in lanes M indicate the presence of methylated alleles. Panel A illustrates HOXA9 (the upper bands represent MSP products, whereas the lower bands are excess primers). Panel B illustrates SCGB3A1 . Abbreviation: Pos, positive control (DNA from normal blood is used as control for unmethylated samples, and in vitro methylated DNA is used as control for methylated samples); Neg, negative control (template replaced by water); Lane 1–6, individual ovarian carcinomas; 100 bp DNA marker from Promega Corp, Madison, WI, USA. The illustration has been processed from a photo including more samples. Hence, the controls have been moved from their original position and pasted adjacent to the selected samples shown here.

    Journal: Molecular Cancer

    Article Title: DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets

    doi: 10.1186/1476-4598-6-45

    Figure Lengend Snippet: Representative methylation-specific polymerase chain reaction (MSP) results from primary ovarian carcinomas . PCR products in lane U indicate the presence of unmethylated alleles whereas PCR products in lanes M indicate the presence of methylated alleles. Panel A illustrates HOXA9 (the upper bands represent MSP products, whereas the lower bands are excess primers). Panel B illustrates SCGB3A1 . Abbreviation: Pos, positive control (DNA from normal blood is used as control for unmethylated samples, and in vitro methylated DNA is used as control for methylated samples); Neg, negative control (template replaced by water); Lane 1–6, individual ovarian carcinomas; 100 bp DNA marker from Promega Corp, Madison, WI, USA. The illustration has been processed from a photo including more samples. Hence, the controls have been moved from their original position and pasted adjacent to the selected samples shown here.

    Article Snippet: Hydroquinone (Sigma Chemical Co., St. Louis, MO, USA) and sodium bisulphite (Sigma Chemical Co, USA) at pH 5.0 were added to the samples to a final concentration of one mM and 3.7 M, respectively, prior to incubation at 50°C for 16 h. Bisulphite treated DNA was purified using the Wizard DNA clean-up kit (Promega Corp, Madison, WI, USA) and eluted in 100 μL MQ water.

    Techniques: Methylation, Polymerase Chain Reaction, Positive Control, In Vitro, Negative Control, Marker

    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