mir172d 1  (New England Biolabs)


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    Name:
    HpaII
    Description:
    HpaII 10 000 units
    Catalog Number:
    r0171l
    Price:
    269
    Size:
    10 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs mir172d 1
    HpaII
    HpaII 10 000 units
    https://www.bioz.com/result/mir172d 1/product/New England Biolabs
    Average 85 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
    mir172d 1 - by Bioz Stars, 2020-04
    85/100 stars

    Images

    1) Product Images from "POWERDRESS and Diversified Expression of the MIR172 Gene Family Bolster the Floral Stem Cell Network"

    Article Title: POWERDRESS and Diversified Expression of the MIR172 Gene Family Bolster the Floral Stem Cell Network

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003218

    Phenotype of the mir172d-1 single mutant and of mir172d-1 in combination with ag-10 and ap2-2 . (A) ag-10 mir172d-1 flower. (B) L er (left) and mir172d-1 (right) siliques. The gynoecia of mir172d-1 occasionally have three fused carpels instead of two. (C) ap2-2 ag-10 mir172d-1 flower. (D) Mature miR172 sequences and the site of the G-to-A mutation in mir172d-1 (indicated by *).
    Figure Legend Snippet: Phenotype of the mir172d-1 single mutant and of mir172d-1 in combination with ag-10 and ap2-2 . (A) ag-10 mir172d-1 flower. (B) L er (left) and mir172d-1 (right) siliques. The gynoecia of mir172d-1 occasionally have three fused carpels instead of two. (C) ap2-2 ag-10 mir172d-1 flower. (D) Mature miR172 sequences and the site of the G-to-A mutation in mir172d-1 (indicated by *).

    Techniques Used: Mutagenesis

    Related Articles

    Clone Assay:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: The Xho I/ Bgl II fragment from this construct was subsequently cloned into the L1-MFGhGFP-1L vector, generating L1−ΔenhMFGhGFP-1L. .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation.

    Blocking Assay:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Homogenised pellets were resuspended in a 100 µl solution of 0.7% low melting point agarose (Promega, Southampton, UK) and spread onto microscope slides pre-coated with 1% standard agarose and the agarose allowed to set by cooling slides on a cold metal block at 4 °C. .. Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons
    Article Snippet: Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100. .. The samples were then loaded onto a preheated ( > 70 °C) PCR block (C1000-Touch, Bio-Rad, Hercules, CA) and subjected to the following cycles of heating and cooling: 98 °C for 3 min, 30× (98 °C for 30 s, 64 °C for 30 s, 72 °C for 210 s) and 72 °C for 10 min.

    Luciferase:

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: .. Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs). .. Transient or stable transfection of DNMT and Wnt7a shRNA was conducted using Sure-Silencing shRNA from SABiosciences or retroviral shRNA from Open Biosystems and Attractene Transfection Reagent (Qiagen).

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain
    Article Snippet: Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs). .. The construction of all luciferase fusion plasmids (pL450, pLup, and pL1.3 k) used in this study has been described previously [ ].

    Stable Transfection:

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs). .. Transient or stable transfection of DNMT and Wnt7a shRNA was conducted using Sure-Silencing shRNA from SABiosciences or retroviral shRNA from Open Biosystems and Attractene Transfection Reagent (Qiagen).

    Construct:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation. .. To determine whether the reactions were carried to completion, following organic extraction and ethanol precipitation, methylated DNA was digested with the methylation-sensitive restriction enzymes Hpa II and Hha I or the methylation-insensitive enzyme Msp I and visualized by electrophoresis on a 0.7% agarose gel.

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain
    Article Snippet: .. Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs). .. Complete methylation was determined by digesting the DNA constructs with the methylation-sensitive restriction enzyme Bstu1, HpaI, HpaII (New England Biolabs) respectly and running the products on an agarose gel.

    Electrophoresis:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively. .. Digested nuclei were exposed to alkali conditions (1 mM Na2 EDTA and 300 mM NaOH) for 20 min at 4 °C, followed by electrophoresis at 300 mA, 0.8 V/cm at 4 °C in the dark for 20 min. After electrophoresis, the slides were neutralised with 0.4 M Tris-HCl buffer, pH 7.5 for 15 min. DNA was fluorescently labelled with a 1:10,000 dilution of SYBR gold for 1 hour (Life Technologies ltd, Paisley, UK).

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation. .. To determine whether the reactions were carried to completion, following organic extraction and ethanol precipitation, methylated DNA was digested with the methylation-sensitive restriction enzymes Hpa II and Hha I or the methylation-insensitive enzyme Msp I and visualized by electrophoresis on a 0.7% agarose gel.

    Article Title: CpG-island promoters drive transcription of human telomeres
    Article Snippet: We prepared genomic DNA with the Wizard genomic DNA kit (Promega) and digested it with HpaII (CpG methylation-sensitive) or MspI (CpG methylation-insensitive) restriction enzymes (New England Biolabs). .. After electrophoresis in 1.2% agarose gels, we denatured DNA, transferred it to nylon membranes, and hybridized it for ∼18 h at 64°C with 32 P-labeled probes generated by random primer labeling of a ∼1-kb DNA fragment comprising 61-29-37 repeats.

    Incubation:

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila
    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. .. The DNA products were incubated at 85°C for 15 min to heat-inactivate the restriction enzymes.

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function
    Article Snippet: Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S). .. After run, the gel was incubated in 0.25 M HCl for 15 min and washed in 0.5 M NaOH, 1.5 M NaCl twice (10 min and 45 min).

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The reconstituted chromatin (total volume = 70 μl) was mixed with 100 μl of a preassembled MNase mixture (94 μl of Ex50 buffer, 5 μl of 100 mM CaCl2 , 1 μl of MNase [50 U/μl; Sigma]) and incubated at room temperature.

    Amplification:

    Article Title: DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation
    Article Snippet: In brief, 1 μg of genomic DNA was digested with HpaII and Msp I (NEB). .. This was followed by PCR amplification and labeling of HpaII and Msp I digestion products.

    Single Cell Gel Electrophoresis:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Paragraph title: Experiment 3 - Methylation sensitive comet assay (MS-CA) ... Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Mass Spectrometry:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Paragraph title: Experiment 3 - Methylation sensitive comet assay (MS-CA) ... Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons
    Article Snippet: DNA methods DNA was isolated from pluripotent stem cells and neurons at indicated times by the salting out method [ ], and the CGG-repeat number in SC120 iPSCs and WCMC37 hESCs and lineages derived from these cells were analyzed by RPT-PCR and MS_RPT-PCR respectively as described before [ ]. .. Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Modification:

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: .. Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs). .. Transient or stable transfection of DNMT and Wnt7a shRNA was conducted using Sure-Silencing shRNA from SABiosciences or retroviral shRNA from Open Biosystems and Attractene Transfection Reagent (Qiagen).

    Derivative Assay:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: The Moloney murine leukemia virus-based construct L1-MFGhGFP-1L which includes the humanized green fluorescent protein ( hGFP )-encoding proviral genome flanked by mouse genomic sequence ( ) and loxP sites, was derived from the proviral vector MFGhGFP ( , ), as described previously ( ). .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation.

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons
    Article Snippet: DNA methods DNA was isolated from pluripotent stem cells and neurons at indicated times by the salting out method [ ], and the CGG-repeat number in SC120 iPSCs and WCMC37 hESCs and lineages derived from these cells were analyzed by RPT-PCR and MS_RPT-PCR respectively as described before [ ]. .. Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Hybridization:

    Article Title: JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis
    Article Snippet: For Southern blots, 5 µg of genomic DNAs was digested with HpaII or MspI (New England Biolabs) overnight. .. The Athila LTR, 5S rDNA repeat, and 180-base-pair (bp) centromere repeat were labeled with α-32 PdCTP for Southern hybridization to determine their DNA methylation status.

    Transfection:

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: Paragraph title: Transfection ... Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs).

    Southern Blot:

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function
    Article Snippet: Paragraph title: Methylation-sensitive Southern Blot ... Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S).

    Ligation:

    Article Title: DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation
    Article Snippet: In brief, 1 μg of genomic DNA was digested with HpaII and Msp I (NEB). .. The digestion products were extracted and then subjected to ligation of HpaII adapter using T4 DNA Ligase.

    Northern Blot:

    Article Title: CpG-island promoters drive transcription of human telomeres
    Article Snippet: We prepared genomic DNA with the Wizard genomic DNA kit (Promega) and digested it with HpaII (CpG methylation-sensitive) or MspI (CpG methylation-insensitive) restriction enzymes (New England Biolabs). .. We performed post-hybridization stringency washes in 0.2× SSC, 0.5% SDS at 64°C, and detected and quantified radioactive signals as for Northern blots.

    Generated:

    Article Title: CpG-island promoters drive transcription of human telomeres
    Article Snippet: We prepared genomic DNA with the Wizard genomic DNA kit (Promega) and digested it with HpaII (CpG methylation-sensitive) or MspI (CpG methylation-insensitive) restriction enzymes (New England Biolabs). .. After electrophoresis in 1.2% agarose gels, we denatured DNA, transferred it to nylon membranes, and hybridized it for ∼18 h at 64°C with 32 P-labeled probes generated by random primer labeling of a ∼1-kb DNA fragment comprising 61-29-37 repeats.

    Sequencing:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: The Moloney murine leukemia virus-based construct L1-MFGhGFP-1L which includes the humanized green fluorescent protein ( hGFP )-encoding proviral genome flanked by mouse genomic sequence ( ) and loxP sites, was derived from the proviral vector MFGhGFP ( , ), as described previously ( ). .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation.

    Fluorescence:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively. .. Comet slides were analysed using a fluorescence microscope (Zeiss, Axiovert) equipped with a 515 to 560 nm excitation filter and a barrier filter of 590 nm. at 400 X magnification using a 40 X oil immersion lens and Komet IV image analysis software (Perceptive Instruments, Bury St Edmunds, UK).

    Methylation:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Paragraph title: Experiment 3 - Methylation sensitive comet assay (MS-CA) ... Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation. .. To determine whether the reactions were carried to completion, following organic extraction and ethanol precipitation, methylated DNA was digested with the methylation-sensitive restriction enzymes Hpa II and Hha I or the methylation-insensitive enzyme Msp I and visualized by electrophoresis on a 0.7% agarose gel.

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs
    Article Snippet: .. To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG). .. An undigested gDNA (5  µ g) served as the negative control.

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain
    Article Snippet: Paragraph title: In vitro methylation of reporter plasmid and reporter gene assays ... Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs).

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila
    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. .. The DNA products were incubated at 85°C for 15 min to heat-inactivate the restriction enzymes.

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function
    Article Snippet: .. Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S). .. The reaction buffer included Cut Smart buffer 1 × (New England Biolabs), spermidine 0.1 M, DTT 0.1 M and 0.25 μl of RNaseH in 50 μl final volume.

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Article Title: CpG-island promoters drive transcription of human telomeres
    Article Snippet: .. We prepared genomic DNA with the Wizard genomic DNA kit (Promega) and digested it with HpaII (CpG methylation-sensitive) or MspI (CpG methylation-insensitive) restriction enzymes (New England Biolabs). .. After electrophoresis in 1.2% agarose gels, we denatured DNA, transferred it to nylon membranes, and hybridized it for ∼18 h at 64°C with 32 P-labeled probes generated by random primer labeling of a ∼1-kb DNA fragment comprising 61-29-37 repeats.

    Isolation:

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons
    Article Snippet: DNA methods DNA was isolated from pluripotent stem cells and neurons at indicated times by the salting out method [ ], and the CGG-repeat number in SC120 iPSCs and WCMC37 hESCs and lineages derived from these cells were analyzed by RPT-PCR and MS_RPT-PCR respectively as described before [ ]. .. Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Labeling:

    Article Title: JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis
    Article Snippet: For Southern blots, 5 µg of genomic DNAs was digested with HpaII or MspI (New England Biolabs) overnight. .. The Athila LTR, 5S rDNA repeat, and 180-base-pair (bp) centromere repeat were labeled with α-32 PdCTP for Southern hybridization to determine their DNA methylation status.

    Article Title: DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation
    Article Snippet: In brief, 1 μg of genomic DNA was digested with HpaII and Msp I (NEB). .. This was followed by PCR amplification and labeling of HpaII and Msp I digestion products.

    Article Title: CpG-island promoters drive transcription of human telomeres
    Article Snippet: We prepared genomic DNA with the Wizard genomic DNA kit (Promega) and digested it with HpaII (CpG methylation-sensitive) or MspI (CpG methylation-insensitive) restriction enzymes (New England Biolabs). .. After electrophoresis in 1.2% agarose gels, we denatured DNA, transferred it to nylon membranes, and hybridized it for ∼18 h at 64°C with 32 P-labeled probes generated by random primer labeling of a ∼1-kb DNA fragment comprising 61-29-37 repeats.

    Polymerase Chain Reaction:

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs
    Article Snippet: Paragraph title: Porcine methylation-specific restriction enzyme PCR assay ... To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG).

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons
    Article Snippet: Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100. .. Total 5 μl of digested DNA from both samples were processed for the repeat PCR with 15-μl PCR reaction mix containing a final composition of 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , 0.2 % Triton X-100, 0.5 μM of the primers Not_FraxC (5′-AGTTCAGCGGCCGCGCTCAGCTCCGTTTCGGTTTCACTTCCGGT-3′) and Not_FraxR4 (5′-CAAGTCGCGGCCGCCTTGTAGAAAGCGCCATTGGAGCCCCGCA-3′), 2.5 M betaine (Sigma-Aldrich), 2 % DMSO (Sigma-Aldrich) and 0.27 mM each dNTP (New England Biolabs), and 0.4 units of Phusion® DNA polymerase (New England Biolabs).

    Article Title: DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation
    Article Snippet: In brief, 1 μg of genomic DNA was digested with HpaII and Msp I (NEB). .. This was followed by PCR amplification and labeling of HpaII and Msp I digestion products.

    Selection:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: After 24 h, 10 whole juvenile daphnids from each treatment and control group were sampled to eliminate tissue selection bias of the Daphnia and/or the presence of eggs in the brood pouch that could lead to potential variations in DNA methylation. .. Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Microscopy:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Homogenised pellets were resuspended in a 100 µl solution of 0.7% low melting point agarose (Promega, Southampton, UK) and spread onto microscope slides pre-coated with 1% standard agarose and the agarose allowed to set by cooling slides on a cold metal block at 4 °C. .. Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Salting Out:

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons
    Article Snippet: DNA methods DNA was isolated from pluripotent stem cells and neurons at indicated times by the salting out method [ ], and the CGG-repeat number in SC120 iPSCs and WCMC37 hESCs and lineages derived from these cells were analyzed by RPT-PCR and MS_RPT-PCR respectively as described before [ ]. .. Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Chromatin Immunoprecipitation:

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila
    Article Snippet: Paragraph title: Chromatin immunoprecipitation and Hpa II methylation assays ... 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.

    Plasmid Preparation:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: The Xho I/ Bgl II fragment from this construct was subsequently cloned into the L1-MFGhGFP-1L vector, generating L1−ΔenhMFGhGFP-1L. .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation.

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: Transfection The reporter plasmid PPAR Response Element luciferase (PPRE) (3 µg), Fzd9 plasmid, and Wnt7a-luciferase were transfected into cells using Lipofectamine Reagent (Invitrogen; Carlsbad, CA, USA). .. Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs).

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain
    Article Snippet: Paragraph title: In vitro methylation of reporter plasmid and reporter gene assays ... Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs).

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The DNA template used for in vitro reconstitution of chromatin was the pBS HPRT 1.8-kb plasmid, which is a pBluescript-derived plasmid containing a 1.8-kb Eco RI-to- Bam HI fragment that includes the entire HPRT promoter. .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier.

    Software:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively. .. Comet slides were analysed using a fluorescence microscope (Zeiss, Axiovert) equipped with a 515 to 560 nm excitation filter and a barrier filter of 590 nm. at 400 X magnification using a 40 X oil immersion lens and Komet IV image analysis software (Perceptive Instruments, Bury St Edmunds, UK).

    Negative Control:

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs
    Article Snippet: To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG). .. An undigested gDNA (5 µ g) served as the negative control.

    shRNA:

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs). .. Transient or stable transfection of DNMT and Wnt7a shRNA was conducted using Sure-Silencing shRNA from SABiosciences or retroviral shRNA from Open Biosystems and Attractene Transfection Reagent (Qiagen).

    Agarose Gel Electrophoresis:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation. .. To determine whether the reactions were carried to completion, following organic extraction and ethanol precipitation, methylated DNA was digested with the methylation-sensitive restriction enzymes Hpa II and Hha I or the methylation-insensitive enzyme Msp I and visualized by electrophoresis on a 0.7% agarose gel.

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs
    Article Snippet: To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG). .. PCR was performed under the following conditions: 94 ∘ C for 5 min, followed by 35 cycles of 94 ∘ C for 30 s, 60 ∘ C for 30 s, and 72 ∘ C for 30 s. The products were electrophoresed on a 2 % ( w / v ) agarose gel in 6X loading buffer (Biosesang, Seongnam, Korea).

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain
    Article Snippet: Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs). .. Complete methylation was determined by digesting the DNA constructs with the methylation-sensitive restriction enzyme Bstu1, HpaI, HpaII (New England Biolabs) respectly and running the products on an agarose gel.

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function
    Article Snippet: Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S). .. The digested DNA was directly loaded on 1% Agarose gel in 1 × TBE.

    In Vitro:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: .. In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation. .. To determine whether the reactions were carried to completion, following organic extraction and ethanol precipitation, methylated DNA was digested with the methylation-sensitive restriction enzymes Hpa II and Hha I or the methylation-insensitive enzyme Msp I and visualized by electrophoresis on a 0.7% agarose gel.

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain
    Article Snippet: Paragraph title: In vitro methylation of reporter plasmid and reporter gene assays ... Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs).

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Ethanol Precipitation:

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation
    Article Snippet: In vitro “partial” methylation of these constructs with Hpa II and Hha I methylases (NEB) was performed simultaneously according to the manufacturer's protocol for Hha I methylation. .. To determine whether the reactions were carried to completion, following organic extraction and ethanol precipitation, methylated DNA was digested with the methylation-sensitive restriction enzymes Hpa II and Hha I or the methylation-insensitive enzyme Msp I and visualized by electrophoresis on a 0.7% agarose gel.

    DNA Methylation Assay:

    Article Title: JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis
    Article Snippet: Paragraph title: DNA methylation analysis ... For Southern blots, 5 µg of genomic DNAs was digested with HpaII or MspI (New England Biolabs) overnight.

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: After 24 h, 10 whole juvenile daphnids from each treatment and control group were sampled to eliminate tissue selection bias of the Daphnia and/or the presence of eggs in the brood pouch that could lead to potential variations in DNA methylation. .. Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs
    Article Snippet: 2.2.2 Porcine methylation-specific restriction enzyme PCR assay The Porcine methylation-specific restriction enzyme PCR (PMP) assay is a PCR-based methylation method that uses methylation-sensitive restriction enzymes to determine DNA methylation status. .. To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG).

    Article Title: DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation
    Article Snippet: Paragraph title: DNA methylation profiling using HELP and custom oligonucleotide arrays ... In brief, 1 μg of genomic DNA was digested with HpaII and Msp I (NEB).

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Article Title: CpG-island promoters drive transcription of human telomeres
    Article Snippet: Paragraph title: DNA methylation analysis ... We prepared genomic DNA with the Wizard genomic DNA kit (Promega) and digested it with HpaII (CpG methylation-sensitive) or MspI (CpG methylation-insensitive) restriction enzymes (New England Biolabs).

    DNA Purification:

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs
    Article Snippet: .. To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG). .. An undigested gDNA (5  µ g) served as the negative control.

    Lysis:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Next, slides were submerged in ice cold lysis buffer containing 10 mM Tris, 100 mM EDTA, 2.5 M NaCl, 10% DMSO and 1% Triton X-100 for 1 h to prepare nucleoids. .. Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer
    Article Snippet: Cells were collected, washed with PBS, and resuspended in Luciferase Reporter Lysis Buffer (Promega). .. Modification of the Wnt7a luciferase was conducted with SssI, HpaII, and HhaI according to the manufacture's protocol (New England Biolabs).

    Activity Assay:

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation
    Article Snippet: Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively. .. Prior to enzymatic digestion, slides were saturated in endonuclease buffer (10 mM Tris–HCl, 10 mM NaCl, 1 mM β-mercaptoethanol and 2 mM EDTA) for 10 min to create optimal conditions for enzyme catalytic activity.

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    New England Biolabs hpy188 iii
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