acii  (New England Biolabs)


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    New England Biolabs acii
    Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and <t>Jemalong.</t> (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of <t>AciI</t> cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.
    Acii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive"

    Article Title: The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-14-174

    Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and Jemalong. (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of AciI cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.
    Figure Legend Snippet: Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and Jemalong. (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of AciI cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.

    Techniques Used: DNA Methylation Assay, Amplification, Methylation, Real-time Polymerase Chain Reaction

    2) Product Images from "Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus"

    Article Title: Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus

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

    doi: 10.1073/pnas.0910955107

    Retrotransposon excision is associated with demethylation of the PvGlo locus. ( A ) Organization of wild-type, transposon, and revertant alleles of PvGlo showing location of upstream methylation-sensitive AciI restriction enzyme sites. The PvGlo -specific primers (F1, R1, R3) and transposon-specific PCR primers (R2, F2) are indicated. The retrotransposon insertion site is shown (-723); LTRs are shown in gray. ( B ) Methylation-sensitive PCR analysis of the wild-type, transposon, and revertant alleles of PvGlo using allele-specific primer combinations as indicated. Primer specificity is demonstrated on DNA from leaves of wild-type (wt) and homozygous Hose in Hose (HH) plants. DNA samples from whorl-1 tissue or combined whorl-2, -3, and -4 tissue from revertant (R), semirevertant (SR), and Hose in Hose (H) flowers from the same plant were either digested with Aci1 or uncut as indicated before PCR amplification using allele-specific primer combinations as shown before fractionation by agarose gel electrophoresis. The different alleles amplified by each primer combination are indicated and the excision allele-specific PCR product is highlighted (*). PCR products generated from uncut genomic DNA that are absent after predigestion with AciI reveal unmethylated sites.
    Figure Legend Snippet: Retrotransposon excision is associated with demethylation of the PvGlo locus. ( A ) Organization of wild-type, transposon, and revertant alleles of PvGlo showing location of upstream methylation-sensitive AciI restriction enzyme sites. The PvGlo -specific primers (F1, R1, R3) and transposon-specific PCR primers (R2, F2) are indicated. The retrotransposon insertion site is shown (-723); LTRs are shown in gray. ( B ) Methylation-sensitive PCR analysis of the wild-type, transposon, and revertant alleles of PvGlo using allele-specific primer combinations as indicated. Primer specificity is demonstrated on DNA from leaves of wild-type (wt) and homozygous Hose in Hose (HH) plants. DNA samples from whorl-1 tissue or combined whorl-2, -3, and -4 tissue from revertant (R), semirevertant (SR), and Hose in Hose (H) flowers from the same plant were either digested with Aci1 or uncut as indicated before PCR amplification using allele-specific primer combinations as shown before fractionation by agarose gel electrophoresis. The different alleles amplified by each primer combination are indicated and the excision allele-specific PCR product is highlighted (*). PCR products generated from uncut genomic DNA that are absent after predigestion with AciI reveal unmethylated sites.

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Fractionation, Agarose Gel Electrophoresis, Generated

    3) Product Images from "Epigenetic regulation of matrix metalloproteinase expression in ameloblastoma"

    Article Title: Epigenetic regulation of matrix metalloproteinase expression in ameloblastoma

    Journal: BMC Clinical Pathology

    doi: 10.1186/1472-6890-12-11

    Representative figure of the methylation analysis. A : Methylation status of MMP-2 in ameloblastoma. M: PCR products when amplified by methylated primers (205 bp); U: PCR products when amplified by unmethylated primers (206 bp); +M: positive control for methylated reaction; +U: positive control for unmethylated reaction. -M and -U: negative controls without DNA. Lines 1 to 3 represent DNA from ameloblastoma samples. B : Methylation status of MMP-9 in ameloblastoma. DNA samples were digested by the AciI restriction enzyme followed by PCR, flanking the restriction sites. Absent band indicates unmethylated profile (U) due to DNA cleavage by the restriction enzyme. Presence of the PCR band represents methylated profile (M) of the MMP-9 gene. +M: methylated positive control; +U: unmethylated positive control; - : negative control without DNA.
    Figure Legend Snippet: Representative figure of the methylation analysis. A : Methylation status of MMP-2 in ameloblastoma. M: PCR products when amplified by methylated primers (205 bp); U: PCR products when amplified by unmethylated primers (206 bp); +M: positive control for methylated reaction; +U: positive control for unmethylated reaction. -M and -U: negative controls without DNA. Lines 1 to 3 represent DNA from ameloblastoma samples. B : Methylation status of MMP-9 in ameloblastoma. DNA samples were digested by the AciI restriction enzyme followed by PCR, flanking the restriction sites. Absent band indicates unmethylated profile (U) due to DNA cleavage by the restriction enzyme. Presence of the PCR band represents methylated profile (M) of the MMP-9 gene. +M: methylated positive control; +U: unmethylated positive control; - : negative control without DNA.

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

    4) Product Images from "Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease"

    Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194036

    Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).
    Figure Legend Snippet: Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

    Techniques Used: Methylation, DNA Methylation Assay, Microarray

    5) Product Images from "Molecular Identification of Mucor and Lichtheimia Species in Pure Cultures of Zygomycetes"

    Article Title: Molecular Identification of Mucor and Lichtheimia Species in Pure Cultures of Zygomycetes

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.35237

    Agarose Gel Electrophoresis of 18sS rRNA PCR Products of Different Mucorals After Restriction Digestion With XmnI and AcII Lanes 12, 11, 10, 9, 8, 2 and 13, M. circinelloides , M. racemosus , M. ramosissimus or M. plumbeus ; Lanes 3, 1, 7, 6, 5, 4, and 14, L. corymbifera or L. blakesleeana ; N, negative control; M, 100 bp molecular size marker.
    Figure Legend Snippet: Agarose Gel Electrophoresis of 18sS rRNA PCR Products of Different Mucorals After Restriction Digestion With XmnI and AcII Lanes 12, 11, 10, 9, 8, 2 and 13, M. circinelloides , M. racemosus , M. ramosissimus or M. plumbeus ; Lanes 3, 1, 7, 6, 5, 4, and 14, L. corymbifera or L. blakesleeana ; N, negative control; M, 100 bp molecular size marker.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Marker

    6) Product Images from "Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species"

    Article Title: Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.10.5999-6007.2005

    Examples of T-RFLP peaks resulting from cow and human fecal samples cut with AciI, MspI, and HaeIII.
    Figure Legend Snippet: Examples of T-RFLP peaks resulting from cow and human fecal samples cut with AciI, MspI, and HaeIII.

    Techniques Used:

    7) Product Images from "High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies"

    Article Title: High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370220968545

    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.
    Figure Legend Snippet: GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.

    Techniques Used: Polymerase Chain Reaction

    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4 . Sample designations identified from Leffler et al. 9
    Figure Legend Snippet: GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4 . Sample designations identified from Leffler et al. 9

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.
    Figure Legend Snippet: Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.

    Techniques Used: Polymerase Chain Reaction, RFLP Assay, Amplification

    8) Product Images from "Serine 1283 in extracellular matrix glycoprotein Reelin is crucial for Reelin’s function in brain development"

    Article Title: Serine 1283 in extracellular matrix glycoprotein Reelin is crucial for Reelin’s function in brain development

    Journal: bioRxiv

    doi: 10.1101/2022.01.06.474241

    Generation of mice expressing Reelin with the S/A 1283 mutation. ( a ) Targeted mutation and genotyping strategies: the wild-type Reelin gene sequence ( rln +/+ ) was used to design zinc finger nucleases 1 and 2 (ZFN1 and ZFN2) which bind close to the codon of serine 1283 (TCA coding for serine). Genomic positions are indicated. In the mutated sequence ( rln A/A ) TCA is replaced by GCG (coding for alanine), thereby generating a restriction site for AciI. Primers P1 and P2 were used to amplify a 530 bp region as indicated. After digestion with AciI, the 530 bp amplicon was cleaved to yield the 199 bp and 331 bp fragments. ( b ) Genotyping of gene-edited mice; P: amplicons with primers P1 and P2; D: AciI-digested amplicon; the pUC vector carrying the donor sequence served as control (donor). ( c ) Southern blot analysis of gene-edited mice; only the 4135 bp EcoRI fragment of the Reelin gene (arrowhead) was detected in two rln +/+ mice and six rln A/A mice using a radiolabeled 530 bp probe derived from PCR amplification using primers P1 and P2 and the pUC vector carrying the donor sequence. See also Supplementary Figure 1. ( d - g ) Phenotype of one-month-old mice expressing Reelin with the S/A 1283 mutation: rln A/A mice display reduced body size ( d ) and body weight ( e ). Mean value + SEM are shown for the body weight (***p
    Figure Legend Snippet: Generation of mice expressing Reelin with the S/A 1283 mutation. ( a ) Targeted mutation and genotyping strategies: the wild-type Reelin gene sequence ( rln +/+ ) was used to design zinc finger nucleases 1 and 2 (ZFN1 and ZFN2) which bind close to the codon of serine 1283 (TCA coding for serine). Genomic positions are indicated. In the mutated sequence ( rln A/A ) TCA is replaced by GCG (coding for alanine), thereby generating a restriction site for AciI. Primers P1 and P2 were used to amplify a 530 bp region as indicated. After digestion with AciI, the 530 bp amplicon was cleaved to yield the 199 bp and 331 bp fragments. ( b ) Genotyping of gene-edited mice; P: amplicons with primers P1 and P2; D: AciI-digested amplicon; the pUC vector carrying the donor sequence served as control (donor). ( c ) Southern blot analysis of gene-edited mice; only the 4135 bp EcoRI fragment of the Reelin gene (arrowhead) was detected in two rln +/+ mice and six rln A/A mice using a radiolabeled 530 bp probe derived from PCR amplification using primers P1 and P2 and the pUC vector carrying the donor sequence. See also Supplementary Figure 1. ( d - g ) Phenotype of one-month-old mice expressing Reelin with the S/A 1283 mutation: rln A/A mice display reduced body size ( d ) and body weight ( e ). Mean value + SEM are shown for the body weight (***p

    Techniques Used: Mouse Assay, Expressing, Mutagenesis, Genotyping Assay, Sequencing, Zinc-Fingers, Amplification, Plasmid Preparation, Southern Blot, Derivative Assay, Polymerase Chain Reaction

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    • acii methylation sensitive restriction endonucleases  (New England Biolabs)
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    New England Biolabs acii methylation sensitive restriction endonucleases
    HpaII and <t>AciI</t> methylation analyses of integrated eGFP in C1 and D1 clones. A) Experimental design showing the amplicon regions and their length within eGFP locus integrated in genomic <t>DNA.</t> HpaII and AciI site are indicated. B) Densitometric analyses of parental C1 clone methylation pattern on eGFP + more positive, eGFP + less positive and eGFP − cells (see also Fig. S9B and Fig. S10B ). ANOVA test gave a statistical significance of p
    Acii Methylation Sensitive Restriction Endonucleases, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/acii methylation sensitive restriction endonucleases/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    HpaII and AciI methylation analyses of integrated eGFP in C1 and D1 clones. A) Experimental design showing the amplicon regions and their length within eGFP locus integrated in genomic DNA. HpaII and AciI site are indicated. B) Densitometric analyses of parental C1 clone methylation pattern on eGFP + more positive, eGFP + less positive and eGFP − cells (see also Fig. S9B and Fig. S10B ). ANOVA test gave a statistical significance of p

    Journal: PLoS ONE

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

    doi: 10.1371/journal.pone.0030851

    Figure Lengend Snippet: HpaII and AciI methylation analyses of integrated eGFP in C1 and D1 clones. A) Experimental design showing the amplicon regions and their length within eGFP locus integrated in genomic DNA. HpaII and AciI site are indicated. B) Densitometric analyses of parental C1 clone methylation pattern on eGFP + more positive, eGFP + less positive and eGFP − cells (see also Fig. S9B and Fig. S10B ). ANOVA test gave a statistical significance of p

    Article Snippet: DNA methylation analysis In order to characterize the relationship between methylation status of eGFP locus and time-dependent eGFP expression, studies by multiplex HpaII /PCR or AciI /PCR were performed on genomic DNA of C1 and D1 modified clones, treated by HpaII or AciI methylation-sensitive restriction endonucleases (New England Biolabs, Ipswich, MA, USA).

    Techniques: Methylation, Amplification

    Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and Jemalong. (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of AciI cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.

    Journal: BMC Plant Biology

    Article Title: The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

    doi: 10.1186/1471-2229-14-174

    Figure Lengend Snippet: Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and Jemalong. (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of AciI cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.

    Article Snippet: To test for differential methylation within MtEIL1 in 2HA and Jemalong e.g. [ ], DNA (10 μg) was digested overnight by AciI (New England Biolabs, http://www.neb.com ) and cleaned through a PCR clean up column (Promega, http://www.promega.com ).

    Techniques: DNA Methylation Assay, Amplification, Methylation, Real-time Polymerase Chain Reaction

    The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

    Journal: PLoS Pathogens

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

    doi: 10.1371/journal.ppat.1003636

    Figure Lengend Snippet: The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

    Article Snippet: 400 U EcoR I-HF, Hind III or Aci I (New England Biolabs) were added to the nuclei and the samples incubated at 37°C overnight with shaking.

    Techniques: Expressing, Ligation, Polymerase Chain Reaction, Amplification

    Alignment of the PTEN gene ( lower ) and pseudogene ( upper ). The region upstream of nucleotide (−841) represents a divergent sequence. MSP primer sets I, II, and III are shown. Set IV-A, -B primers were used for MSRA to amplify the PTEN gene and pseudogene, respectively. Aci I restriction sites are indicated with triangles. Differences between the two sequences are shown with the pseudogene sequence on top for MSRA are shown. Genomic position is defined by the location relative to the translational start site of PTEN (GenBank accession number AF143312).

    Journal: The American Journal of Pathology

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene

    doi:

    Figure Lengend Snippet: Alignment of the PTEN gene ( lower ) and pseudogene ( upper ). The region upstream of nucleotide (−841) represents a divergent sequence. MSP primer sets I, II, and III are shown. Set IV-A, -B primers were used for MSRA to amplify the PTEN gene and pseudogene, respectively. Aci I restriction sites are indicated with triangles. Differences between the two sequences are shown with the pseudogene sequence on top for MSRA are shown. Genomic position is defined by the location relative to the translational start site of PTEN (GenBank accession number AF143312).

    Article Snippet: Three hundred nanograms of genomic cell line DNA was incubated with 10 units of Aci I (New England Biolabs) at 37°C for 16 hours.

    Techniques: Sequencing

    Methylation-specific PCR using set III PTEN specific primers ( a ) and methylation-sensitive restriction analysis ( b, c, d ) using primer set IV-A ( PTEN ) respectively for cell lines. MSP analysis, as well as MSRA using primer set IV-A ( PTEN ), show lack of methylation in cell lines ( a, b ). Results for MSRA using set IV-B and -C primers ( c, d ) show methylation positive products in the same cell lines. NL treated with Sss I methyltransferase was used as a methylated positive control. Following Aci I digestion, equal amounts of cell line DNA were used for PCR analysis. U, unmethylated; M, methylated; N, undigested DNA; D, digested DNA; NL, normal lymphocyte DNA.

    Journal: The American Journal of Pathology

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene

    doi:

    Figure Lengend Snippet: Methylation-specific PCR using set III PTEN specific primers ( a ) and methylation-sensitive restriction analysis ( b, c, d ) using primer set IV-A ( PTEN ) respectively for cell lines. MSP analysis, as well as MSRA using primer set IV-A ( PTEN ), show lack of methylation in cell lines ( a, b ). Results for MSRA using set IV-B and -C primers ( c, d ) show methylation positive products in the same cell lines. NL treated with Sss I methyltransferase was used as a methylated positive control. Following Aci I digestion, equal amounts of cell line DNA were used for PCR analysis. U, unmethylated; M, methylated; N, undigested DNA; D, digested DNA; NL, normal lymphocyte DNA.

    Article Snippet: Three hundred nanograms of genomic cell line DNA was incubated with 10 units of Aci I (New England Biolabs) at 37°C for 16 hours.

    Techniques: Methylation, Polymerase Chain Reaction, Positive Control