hpa ii New England Biolabs Search Results


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  • 99
    New England Biolabs hpa ii
    P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the <t>DNA</t> methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either <t>Hpa</t> II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P
    Hpa Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase
    P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the <t>DNA</t> methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either <t>Hpa</t> II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs msp i
    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme <t>Msp</t> I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated <t>CpG</t> retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
    Msp I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1457 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase
    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of <t>T4</t> DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hha i
    Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with <t>Hpa</t> II, <t>Hha</t> I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).
    Hha I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 800 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bst ui
    Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic <t>DNA</t> or DNA digested with Hpa II, Hha I or <t>Bst</t> UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).
    Bst Ui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mcrbc
    Comparison of HM-PCR and HM-WGA-PCR results for eleven CGIs . Genomic DNA from peripheral blood leukocytes was digested with Mock (lane 1), <t>Hpa</t> II or Hha I (lane 2), Msp I (lane 3), or <t>McrBC</t> (lane 4). The digested genomic DNA were used for PCR amplification either directly (left; HM-PCR) or after whole-genome-amplification (right; HM-WGA-PCR) using primer pairs for the eight CGIs on chromosome 21 (A~D) and three CGIs on chromosome 11 (E). Here, when Hha I-digested genomic DNA was used in lane 2, 1 ul of distilled water was used in lane 3 in place of Msp I-digested genomic DNA. PCR products were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. Results of bisulfite sequencing are shown for the eight CGIs on chromosome 21 (A~D). Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Each row of circles represents each sequenced clone of bisufite PCR products.
    Mcrbc, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs s adenosylmethionine
    Comparison of HM-PCR and HM-WGA-PCR results for eleven CGIs . Genomic DNA from peripheral blood leukocytes was digested with Mock (lane 1), <t>Hpa</t> II or Hha I (lane 2), Msp I (lane 3), or <t>McrBC</t> (lane 4). The digested genomic DNA were used for PCR amplification either directly (left; HM-PCR) or after whole-genome-amplification (right; HM-WGA-PCR) using primer pairs for the eight CGIs on chromosome 21 (A~D) and three CGIs on chromosome 11 (E). Here, when Hha I-digested genomic DNA was used in lane 2, 1 ul of distilled water was used in lane 3 in place of Msp I-digested genomic DNA. PCR products were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. Results of bisulfite sequencing are shown for the eight CGIs on chromosome 21 (A~D). Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Each row of circles represents each sequenced clone of bisufite PCR products.
    S Adenosylmethionine, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 868 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs hpa ii methyltransferase
    The Klf1 exon 2 CGI is unmethylated during development. A ) Schematic of the mouse Klf1 gene across exon 2 and the flanking introns shows salient restriction enzyme sites within the region and a 1.1-kbp cDNA probe used for Southern blot. B ) Genomic DNA samples from whole testis between P5 and P45 (lanes 1–7, left and center panels) and P45 spleen (lane 8). DNAs were purified and double-digested with Eco RI and Bam HI restriction enzymes to liberate the 1.85-kb genomic fragment containing the Klf1 exon 2 CGI. Thereafter, the DNA samples were further digested with <t>Hpa</t> II (left panel) or not digested (center panel) and then Southern blotted using the 1.1-kb probe shown in A . In addition, several samples were methylated using Hpa II <t>-methyltransferase</t> then digested with Hpa II (right panel) and Southern blotted. Because of the total number of samples in this experiment, three Southern blots were generated, and the relevant lanes were used to form composite panels. Data for the P5–P15 lanes (break in Southern blots) were obtained from separate Southern blots. Arrowheads in A , locations of Hpa II restriction sites; FL in B , full-length 1.85-kb Eco RI - Bam HI fragments that are visible in the center and right panels; Spl, spleen.
    Hpa Ii Methyltransferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cutsmart buffer
    The Klf1 exon 2 CGI is unmethylated during development. A ) Schematic of the mouse Klf1 gene across exon 2 and the flanking introns shows salient restriction enzyme sites within the region and a 1.1-kbp cDNA probe used for Southern blot. B ) Genomic DNA samples from whole testis between P5 and P45 (lanes 1–7, left and center panels) and P45 spleen (lane 8). DNAs were purified and double-digested with Eco RI and Bam HI restriction enzymes to liberate the 1.85-kb genomic fragment containing the Klf1 exon 2 CGI. Thereafter, the DNA samples were further digested with <t>Hpa</t> II (left panel) or not digested (center panel) and then Southern blotted using the 1.1-kb probe shown in A . In addition, several samples were methylated using Hpa II <t>-methyltransferase</t> then digested with Hpa II (right panel) and Southern blotted. Because of the total number of samples in this experiment, three Southern blots were generated, and the relevant lanes were used to form composite panels. Data for the P5–P15 lanes (break in Southern blots) were obtained from separate Southern blots. Arrowheads in A , locations of Hpa II restriction sites; FL in B , full-length 1.85-kb Eco RI - Bam HI fragments that are visible in the center and right panels; Spl, spleen.
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    New England Biolabs ecor i
    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with <t>EcoR</t> I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
    Ecor I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hpa ii
    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for <t>Msp</t> I/ <t>Hpa</t> II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.
    Hpa Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cpg methyltransferase
    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for <t>Msp</t> I/ <t>Hpa</t> II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.
    Cpg Methyltransferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dde i
    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for <t>Msp</t> I/ <t>Hpa</t> II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.
    Dde I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen dneasy plant mini kit
    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for <t>Msp</t> I/ <t>Hpa</t> II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.
    Dneasy Plant Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 19981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hpa ii restriction endonuclease
    Resistance of methylated B. bovis DNA to cleavage by <t>Hpa</t> II. DNAs prepared from E. coli or B. bovis were treated with <t>Sss</t> I methylase and compared with untreated DNA for sensitivity to Hpa II digestion. DNA was visualized after electrophoresis on ethidium bromide-stained agarose gels. Lanes: M, 1-kb markers; 1, untreated DNA; 2, untreated DNA incubated with Hpa II; 3, methylated DNA; 4, methylated DNA incubated with Hpa II.
    Hpa Ii Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mse i
    Overall strategy for cloning methylated CpG islands. In step 1, genomic <t>DNA</t> was digested with <t>Mse</t> I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
    Mse I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna polymerase
    Overall strategy for cloning methylated CpG islands. In step 1, genomic <t>DNA</t> was digested with <t>Mse</t> I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
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    Millipore 1x t4 dna ligase buffer
    Overall strategy for cloning methylated CpG islands. In step 1, genomic <t>DNA</t> was digested with <t>Mse</t> I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
    1x T4 Dna Ligase Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs aci i
    Overall strategy for cloning methylated CpG islands. In step 1, genomic <t>DNA</t> was digested with <t>Mse</t> I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
    Aci I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen allprep dna rna mini kit
    Overall strategy for cloning methylated CpG islands. In step 1, genomic <t>DNA</t> was digested with <t>Mse</t> I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
    Allprep Dna Rna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 7493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs blunt ta ligase master mix
    Overall strategy for cloning methylated CpG islands. In step 1, genomic <t>DNA</t> was digested with <t>Mse</t> I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
    Blunt Ta Ligase Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the DNA methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P

    Journal: Disease Models & Mechanisms

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

    doi: 10.1242/dmm.022350

    Figure Lengend Snippet: P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the DNA methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P

    Article Snippet: To perform the Hpa II methylation assay, genomic DNA was extracted from cells, followed by digestion with 2 units of Hpa II or Msp I (New England Biolabs) for 4 h at 37°C.

    Techniques: Expressing, Inhibition, LDH Cytotoxicity Assay, Concentration Assay, Activity Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Amplification, Western Blot, Mutagenesis

    Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P

    Journal: Disease Models & Mechanisms

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

    doi: 10.1242/dmm.022350

    Figure Lengend Snippet: Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P

    Article Snippet: To perform the Hpa II methylation assay, genomic DNA was extracted from cells, followed by digestion with 2 units of Hpa II or Msp I (New England Biolabs) for 4 h at 37°C.

    Techniques: Expressing, Sequencing, In Vitro, Binding Assay, Polymerase Chain Reaction, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, Construct, Chromatin Immunoprecipitation, DNA Methylation Assay, Plasmid Preparation, Methylation, Amplification, Activity Assay

    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Journal: BMC Molecular Biology

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles

    doi: 10.1186/s12867-017-0083-2

    Figure Lengend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Article Snippet: Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA).

    Techniques: Methylation, Ligation, Polymerase Chain Reaction, Amplification

    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Journal: BMC Molecular Biology

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles

    doi: 10.1186/s12867-017-0083-2

    Figure Lengend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Article Snippet: Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA).

    Techniques: Methylation, Ligation, Polymerase Chain Reaction, Amplification

    Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with Hpa II, Hha I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).

    Journal: PLoS ONE

    Article Title: 5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation

    doi: 10.1371/journal.pone.0103714

    Figure Lengend Snippet: Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with Hpa II, Hha I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).

    Article Snippet: Digestion with methylation-sensitive restriction enzymes Genomic DNA (500 ng) was digested with Hpa II (Invitrogen, Carlsbad, CA, USA), Bst UI and Hha I (New England Biolabs, Ipswich, MA, USA) for 6 h at 37°C (Hpa II and Hha I) or 60°C (Bst UI), or was mock-digested without the restriction enzymes.

    Techniques: Methylation, Generated, Polymerase Chain Reaction, Fluorescence

    Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with Hpa II, Hha I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).

    Journal: PLoS ONE

    Article Title: 5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation

    doi: 10.1371/journal.pone.0103714

    Figure Lengend Snippet: Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with Hpa II, Hha I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).

    Article Snippet: Digestion with methylation-sensitive restriction enzymes Genomic DNA (500 ng) was digested with Hpa II (Invitrogen, Carlsbad, CA, USA), Bst UI and Hha I (New England Biolabs, Ipswich, MA, USA) for 6 h at 37°C (Hpa II and Hha I) or 60°C (Bst UI), or was mock-digested without the restriction enzymes.

    Techniques: Methylation, Generated, Polymerase Chain Reaction, Fluorescence

    Comparison of HM-PCR and HM-WGA-PCR results for eleven CGIs . Genomic DNA from peripheral blood leukocytes was digested with Mock (lane 1), Hpa II or Hha I (lane 2), Msp I (lane 3), or McrBC (lane 4). The digested genomic DNA were used for PCR amplification either directly (left; HM-PCR) or after whole-genome-amplification (right; HM-WGA-PCR) using primer pairs for the eight CGIs on chromosome 21 (A~D) and three CGIs on chromosome 11 (E). Here, when Hha I-digested genomic DNA was used in lane 2, 1 ul of distilled water was used in lane 3 in place of Msp I-digested genomic DNA. PCR products were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. Results of bisulfite sequencing are shown for the eight CGIs on chromosome 21 (A~D). Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Each row of circles represents each sequenced clone of bisufite PCR products.

    Journal: BMC Research Notes

    Article Title: Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

    doi: 10.1186/1756-0500-4-179

    Figure Lengend Snippet: Comparison of HM-PCR and HM-WGA-PCR results for eleven CGIs . Genomic DNA from peripheral blood leukocytes was digested with Mock (lane 1), Hpa II or Hha I (lane 2), Msp I (lane 3), or McrBC (lane 4). The digested genomic DNA were used for PCR amplification either directly (left; HM-PCR) or after whole-genome-amplification (right; HM-WGA-PCR) using primer pairs for the eight CGIs on chromosome 21 (A~D) and three CGIs on chromosome 11 (E). Here, when Hha I-digested genomic DNA was used in lane 2, 1 ul of distilled water was used in lane 3 in place of Msp I-digested genomic DNA. PCR products were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. Results of bisulfite sequencing are shown for the eight CGIs on chromosome 21 (A~D). Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Each row of circles represents each sequenced clone of bisufite PCR products.

    Article Snippet: HM-WGA-PCR Genomic DNA (500 ng) was digested with 50 U of Hpa II, Hha I, Msp I (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers.

    Techniques: Polymerase Chain Reaction, Whole Genome Amplification, Amplification, Staining, Methylation Sequencing, Methylation

    Principles of the Hpa II- McrBC whole-genome-amplification PCR (HM-WGA-PCR) method . The two parallel lines in the

    Journal: BMC Research Notes

    Article Title: Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

    doi: 10.1186/1756-0500-4-179

    Figure Lengend Snippet: Principles of the Hpa II- McrBC whole-genome-amplification PCR (HM-WGA-PCR) method . The two parallel lines in the "Digestion" panel indicate genomic amplicons from both alleles. The circled "m" indicates a methylated CpG dinucleotide. Hpa II digests unmethylated CCGG, but not methylated C m CGG. In contrast, McrBC digests methylated R m C 40-80 R m C sequences, but not unmethylated RC 40-80 RC. Genomic DNA is digested with Hpa II and McrBC independently. Subsequently, an aliquot of each restriction-enzyme-digested DNA (50 ng) is subjected to whole-genome-amplification (WGA) to yield 5 μg of whole-genome-amplified DNA. Using an aliquot of the amplified DNA (50 ng), the target DNA region is PCR-amplified by the primer pair (dotted arrows). The PCR products from the Hpa II/ Hha I-digested and McrBC -digested DNA are electrophoresed, stained with ethidium bromide, and visualized by UV illumination. If an amplicon is fully methylated (i.e., complete methylation), it is digested by McrBC , but not by Hpa II. Thus, it is amplified from the Hpa II-digested and whole-genome-amplified DNA, but not from the McrBC -digested and whole-genome-amplified DNA. By contrast, if an amplicon totally escapes methylation (i.e., null methylation), it is digested by Hpa II, but not by McrBC . Thus, it is amplified from McrBC -digested and whole-genome-amplified DNA, but not from Hpa II-digested and whole-genome-amplified DNA. If an amplicon contains both methylated and unmethylated alleles (i.e., composite methylation), it is amplified from both whole-genome-amplified templates. If an amplicon is partially methylated on both alleles (i.e., incomplete methylation), it is amplified from neither whole-genome-amplified template.

    Article Snippet: HM-WGA-PCR Genomic DNA (500 ng) was digested with 50 U of Hpa II, Hha I, Msp I (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers.

    Techniques: Whole Genome Amplification, Polymerase Chain Reaction, Methylation, Amplification, Staining

    The Klf1 exon 2 CGI is unmethylated during development. A ) Schematic of the mouse Klf1 gene across exon 2 and the flanking introns shows salient restriction enzyme sites within the region and a 1.1-kbp cDNA probe used for Southern blot. B ) Genomic DNA samples from whole testis between P5 and P45 (lanes 1–7, left and center panels) and P45 spleen (lane 8). DNAs were purified and double-digested with Eco RI and Bam HI restriction enzymes to liberate the 1.85-kb genomic fragment containing the Klf1 exon 2 CGI. Thereafter, the DNA samples were further digested with Hpa II (left panel) or not digested (center panel) and then Southern blotted using the 1.1-kb probe shown in A . In addition, several samples were methylated using Hpa II -methyltransferase then digested with Hpa II (right panel) and Southern blotted. Because of the total number of samples in this experiment, three Southern blots were generated, and the relevant lanes were used to form composite panels. Data for the P5–P15 lanes (break in Southern blots) were obtained from separate Southern blots. Arrowheads in A , locations of Hpa II restriction sites; FL in B , full-length 1.85-kb Eco RI - Bam HI fragments that are visible in the center and right panels; Spl, spleen.

    Journal: Biology of Reproduction

    Article Title: Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice 1

    doi: 10.1095/biolreprod.112.099879

    Figure Lengend Snippet: The Klf1 exon 2 CGI is unmethylated during development. A ) Schematic of the mouse Klf1 gene across exon 2 and the flanking introns shows salient restriction enzyme sites within the region and a 1.1-kbp cDNA probe used for Southern blot. B ) Genomic DNA samples from whole testis between P5 and P45 (lanes 1–7, left and center panels) and P45 spleen (lane 8). DNAs were purified and double-digested with Eco RI and Bam HI restriction enzymes to liberate the 1.85-kb genomic fragment containing the Klf1 exon 2 CGI. Thereafter, the DNA samples were further digested with Hpa II (left panel) or not digested (center panel) and then Southern blotted using the 1.1-kb probe shown in A . In addition, several samples were methylated using Hpa II -methyltransferase then digested with Hpa II (right panel) and Southern blotted. Because of the total number of samples in this experiment, three Southern blots were generated, and the relevant lanes were used to form composite panels. Data for the P5–P15 lanes (break in Southern blots) were obtained from separate Southern blots. Arrowheads in A , locations of Hpa II restriction sites; FL in B , full-length 1.85-kb Eco RI - Bam HI fragments that are visible in the center and right panels; Spl, spleen.

    Article Snippet: Genomic DNAs purified from testes were restriction digested with Eco RI and Bam HI (New England Biolabs) at 37°C for 4–6 h. DNA samples were then either restriction digested with Hpa II (New England Biolabs) for 4–6 h or methylated in vitro using Hpa II methyltransferase (New England Biolabs) and digested with Hpa II .

    Techniques: Southern Blot, Purification, Methylation, Generated

    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Journal: International Journal of Molecular Sciences

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    doi: 10.3390/ijms17101690

    Figure Lengend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Article Snippet: Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method.

    Techniques: DNA Methylation Assay, Methylation

    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.

    Journal: Scientific Reports

    Article Title: Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)

    doi: 10.1038/s41598-017-07231-7

    Figure Lengend Snippet: Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.

    Article Snippet: Southern and dot blot hybridisation Genomic DNAs were digested with Hae III (Fermentas), Hpa II (Fermentas), Msp I (Promega), Pvu II (New England Biolabs) and Taq I (Roche).

    Techniques: Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification

    Resistance of methylated B. bovis DNA to cleavage by Hpa II. DNAs prepared from E. coli or B. bovis were treated with Sss I methylase and compared with untreated DNA for sensitivity to Hpa II digestion. DNA was visualized after electrophoresis on ethidium bromide-stained agarose gels. Lanes: M, 1-kb markers; 1, untreated DNA; 2, untreated DNA incubated with Hpa II; 3, methylated DNA; 4, methylated DNA incubated with Hpa II.

    Journal: Infection and Immunity

    Article Title: DNA and a CpG Oligonucleotide Derived from Babesia bovis Are Mitogenic for Bovine B Cells

    doi:

    Figure Lengend Snippet: Resistance of methylated B. bovis DNA to cleavage by Hpa II. DNAs prepared from E. coli or B. bovis were treated with Sss I methylase and compared with untreated DNA for sensitivity to Hpa II digestion. DNA was visualized after electrophoresis on ethidium bromide-stained agarose gels. Lanes: M, 1-kb markers; 1, untreated DNA; 2, untreated DNA incubated with Hpa II; 3, methylated DNA; 4, methylated DNA incubated with Hpa II.

    Article Snippet: CpG methylase ( Sss I methylase) and Hpa II restriction endonuclease were purchased from New England BioLabs (Beverly, Mass.), and methylation was performed according to the manufacturer’s instructions.

    Techniques: Methylation, Electrophoresis, Staining, Incubation

    Overall strategy for cloning methylated CpG islands. In step 1, genomic DNA was digested with Mse I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.

    Journal: Genome Research

    Article Title: A Genome-Wide Screen for Normally Methylated Human CpG Islands That Can Identify Novel Imprinted Genes

    doi: 10.1101/gr.224102

    Figure Lengend Snippet: Overall strategy for cloning methylated CpG islands. In step 1, genomic DNA was digested with Mse I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.

    Article Snippet: Genomic DNA was digested with Mse I alone or Mse I together with a methylcytosine-sensitive (Hpa II, LTI, or Sma I, NEB) or methyl-insensitive (Msp I or Xma I, NEB) restriction endonuclease according to the manufacturer’s conditions.

    Techniques: Clone Assay, Methylation, Transformation Assay