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    FspEI
    Description:
    FspEI 200 units
    Catalog Number:
    r0662s
    Price:
    110
    Size:
    200 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs fspei
    FspEI
    FspEI 200 units
    https://www.bioz.com/result/fspei/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    fspei - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI"

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

    Journal: Genome Research

    doi: 10.1101/gr.222885.117

    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).
    Figure Legend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Techniques Used: Ligation, Fractionation, Amplification, Sequencing

    2) Product Images from "MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes"

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

    Journal: Open Biology

    doi: 10.1098/rsob.150130

    Schematic overview of the procedure for MethylRAD library preparation. Genomic DNA is digested with the restriction enzyme FspEI, producing 32-bp fragments including four-base 3′ overhangs. Adaptors with compatible overhangs (NNNN) are ligated to each end of these fragments. Tag density can be adjusted using adaptors with selective overhangs (e.g. NNNG). The constructs are amplified and purified by gel extraction. Sample-specific barcodes are incorporated in each construct by PCR, and the products pooled for sequencing.
    Figure Legend Snippet: Schematic overview of the procedure for MethylRAD library preparation. Genomic DNA is digested with the restriction enzyme FspEI, producing 32-bp fragments including four-base 3′ overhangs. Adaptors with compatible overhangs (NNNN) are ligated to each end of these fragments. Tag density can be adjusted using adaptors with selective overhangs (e.g. NNNG). The constructs are amplified and purified by gel extraction. Sample-specific barcodes are incorporated in each construct by PCR, and the products pooled for sequencing.

    Techniques Used: Construct, Amplification, Purification, Gel Extraction, Polymerase Chain Reaction, Sequencing

    3) Product Images from "A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI"

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-015-0139-7

    Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.
    Figure Legend Snippet: Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.

    Techniques Used: Sequencing, Construct

    4) Product Images from "Novel features of telomere biology revealed by the absence of telomeric DNA methylation"

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation

    Journal: Genome Research

    doi: 10.1101/gr.202465.115

    Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.
    Figure Legend Snippet: Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.

    Techniques Used: Methylation, DNA Methylation Assay, Sequencing, Southern Blot, Hybridization, Staining

    Related Articles

    Electrophoresis:

    Article Title: A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation
    Article Snippet: To identify m5C methylation, gDNA was treated with the methylation-sensitive REases McrBC, FspEI, and MspJI (NEB). .. A total of 500 ng gDNA was incubated with each REase at 37°C for 3 h (McrBC) or 6 h (FspEI and MspJI), followed by analysis by electrophoresis on a 1% agarose gel with ethidium bromide.

    Negative Control:

    Article Title: Heritable Epigenetic Variation among Maize Inbreds
    Article Snippet: As expected, the IP negative control and qPCR no template controls either did not amplify or amplified approximately 10 cycles after the experimental samples ( > 1000 fold difference, data not shown). .. 1 microgram of genomic DNA was digested for 16 hr with MspJI or FspEI (New England Biolabs).

    Amplification:

    Article Title: Heritable Epigenetic Variation among Maize Inbreds
    Article Snippet: As expected, the IP negative control and qPCR no template controls either did not amplify or amplified approximately 10 cycles after the experimental samples ( > 1000 fold difference, data not shown). .. 1 microgram of genomic DNA was digested for 16 hr with MspJI or FspEI (New England Biolabs).

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA). .. Then, two adaptors were added to the digested DNAs by T4 DNA ligase (NEB, USA), and the ligation products were amplified by specific primers.

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes
    Article Snippet: MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion. .. Ligation products were amplified in 20 µl reactions containing 7 µl ligated DNA, 0.2 µM of each primer (p1 and p2), 0.3 mM dNTP, 1 × Phusion HF buffer and 0.4 U Phusion high-fidelity DNA polymerase (NEB).

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. Subsequently, amplification of the enzyme-treated samples and paired untreated samples was carried out using specific primers that encompassed the target MspJI/FspEI site.

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI
    Article Snippet: .. Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol. ..

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: .. The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions. .. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and were subjected to single end sequencing on an Illumina HiSeq2500 sequencer.

    Agarose Gel Electrophoresis:

    Article Title: A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation
    Article Snippet: To identify m5C methylation, gDNA was treated with the methylation-sensitive REases McrBC, FspEI, and MspJI (NEB). .. A total of 500 ng gDNA was incubated with each REase at 37°C for 3 h (McrBC) or 6 h (FspEI and MspJI), followed by analysis by electrophoresis on a 1% agarose gel with ethidium bromide.

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes
    Article Snippet: .. MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion. .. Then, 10 µl ligation master mix containing 0.2 µM each of two adaptors, 1 mM ATP and 800 U of T4 DNA ligase (NEB) was added to the digestion solution, and incubated for 6–12 h at 4°C.

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation
    Article Snippet: Equal amounts of genomic DNA (500 ng) were undigested or digested with the restriction enzymes HpaII, FspEI, or McrBC following the supplier's instructions (New England Biolabs). .. The resulting DNA samples were purified, further digested with Tru9I, resolved on an agarose gel, transferred to a Hybond-N+ membrane, and hybridized with a telomeric probe, as previously described ( ).

    Article Title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A
    Article Snippet: A total of 1 µg mtDNA isolated from wild-type and DNMT3A -KO HEK239T cells was digested in parallel using DNA methylation–dependent restriction enzyme FspEI (NEB, R0662S), which recognizes the Cm C site (the second cytosine can be in the context of CG, CHG, or CHH). .. After digestion, the resulting mtDNA was subjected to agarose gel electrophoresis.

    High Throughput Screening Assay:

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: Paragraph title: MethylRAD library construction and high-throughput sequencing ... Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA).

    Methylation:

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: .. Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA). .. FspEI can recognize 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the Cm CGG and m CHG sites, and generate a double-stranded DNA break on the 30 side of the modified cytosine at a fixed distance (N12 /N16 ).

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: Paragraph title: Methylation digest. ... One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture.

    Article Title: A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation
    Article Snippet: .. To identify m5C methylation, gDNA was treated with the methylation-sensitive REases McrBC, FspEI, and MspJI (NEB). .. A total of 500 ng gDNA was incubated with each REase at 37°C for 3 h (McrBC) or 6 h (FspEI and MspJI), followed by analysis by electrophoresis on a 1% agarose gel with ethidium bromide.

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: .. In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation
    Article Snippet: Paragraph title: Methylation-dependent restriction enzyme analyses of telomeric DNA methylation ... Equal amounts of genomic DNA (500 ng) were undigested or digested with the restriction enzymes HpaII, FspEI, or McrBC following the supplier's instructions (New England Biolabs).

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions. .. The methyl-RAD methylated tag libraries were constructed using the MethylRAD technique and sequenced on the Hiseq X Ten platform.

    Article Title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A
    Article Snippet: .. A total of 1 µg mtDNA isolated from wild-type and DNMT3A -KO HEK239T cells was digested in parallel using DNA methylation–dependent restriction enzyme FspEI (NEB, R0662S), which recognizes the Cm C site (the second cytosine can be in the context of CG, CHG, or CHH). .. A similar experiment was performed by digesting the same amount of mtDNA with DNA methylation–independent restriction enzyme BstNI (NEB, R0168S), which recognizes the CCWGG (W = A or T) site.

    Isolation:

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: Paragraph title: DNA sample isolation and MethylRAD library preparation and sequencing ... The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions.

    Article Title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A
    Article Snippet: .. A total of 1 µg mtDNA isolated from wild-type and DNMT3A -KO HEK239T cells was digested in parallel using DNA methylation–dependent restriction enzyme FspEI (NEB, R0662S), which recognizes the Cm C site (the second cytosine can be in the context of CG, CHG, or CHH). .. A similar experiment was performed by digesting the same amount of mtDNA with DNA methylation–independent restriction enzyme BstNI (NEB, R0168S), which recognizes the CCWGG (W = A or T) site.

    Ligation:

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA). .. Then, two adaptors were added to the digested DNAs by T4 DNA ligase (NEB, USA), and the ligation products were amplified by specific primers.

    Article Title: What drives phenotypic divergence among coral clonemates of Acropora palmata?. What drives phenotypic divergence among coral clonemates of Acropora palmata?
    Article Snippet: A total of 100 ng of HMW genomic DNA was digested with Fsp EI (NEB) at 37°C for 4 hr. .. Ligation was performed in a 30‐µl reaction with 20 µl of digested DNA, 0.2 µm each of two adaptors, 1 mm ATP and 800 U of T4 DNA ligase (NEB).

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes
    Article Snippet: MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion. .. Then, 10 µl ligation master mix containing 0.2 µM each of two adaptors, 1 mM ATP and 800 U of T4 DNA ligase (NEB) was added to the digestion solution, and incubated for 6–12 h at 4°C.

    Sequencing:

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: Paragraph title: MethylRAD library construction and high-throughput sequencing ... Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA).

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture. .. Samples were digested for 2 h at 37°C and then heat inactivated at 80°C for 15 min. For coverage analysis, rarefaction analysis was performed on Illumina MiSeq sequencing reads derived from samples digested with MspJI or FspEI (digest and SWGA) or mock digested with no enzyme prior to SWGA with primer set pvset1920.

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes
    Article Snippet: .. MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion. .. Then, 10 µl ligation master mix containing 0.2 µM each of two adaptors, 1 mM ATP and 800 U of T4 DNA ligase (NEB) was added to the digestion solution, and incubated for 6–12 h at 4°C.

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: .. In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: Paragraph title: DNA sample isolation and MethylRAD library preparation and sequencing ... The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions.

    Construct:

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: 150–200 ng of DNAs were used to construct the MethylRAD library with slight modifications [ ]. .. Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA).

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions. .. The methyl-RAD methylated tag libraries were constructed using the MethylRAD technique and sequenced on the Hiseq X Ten platform.

    DNA Methylation Assay:

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation
    Article Snippet: Paragraph title: Methylation-dependent restriction enzyme analyses of telomeric DNA methylation ... Equal amounts of genomic DNA (500 ng) were undigested or digested with the restriction enzymes HpaII, FspEI, or McrBC following the supplier's instructions (New England Biolabs).

    SYBR Green Assay:

    Article Title: Heritable Epigenetic Variation among Maize Inbreds
    Article Snippet: Mez1 qPCR reactions were conducted using 100 ng DNA and Light Cycler480 SYBR Green I Master (Roche, Cat # 04707516001) on the LightCycler480 instrument (Roche) in accordance with Roche's protocol for SYBR Green on the LightCycler480. .. 1 microgram of genomic DNA was digested for 16 hr with MspJI or FspEI (New England Biolabs).

    Acrylamide Gel Assay:

    Article Title: What drives phenotypic divergence among coral clonemates of Acropora palmata?. What drives phenotypic divergence among coral clonemates of Acropora palmata?
    Article Snippet: A total of 100 ng of HMW genomic DNA was digested with Fsp EI (NEB) at 37°C for 4 hr. .. Five microlitres of digested DNA was run on an acrylamide gel to verify digestion.

    Incubation:

    Article Title: What drives phenotypic divergence among coral clonemates of Acropora palmata?. What drives phenotypic divergence among coral clonemates of Acropora palmata?
    Article Snippet: Time of incubation in extraction buffer was increased to 16–20 hr. .. A total of 100 ng of HMW genomic DNA was digested with Fsp EI (NEB) at 37°C for 4 hr.

    Article Title: A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation
    Article Snippet: To identify m5C methylation, gDNA was treated with the methylation-sensitive REases McrBC, FspEI, and MspJI (NEB). .. A total of 500 ng gDNA was incubated with each REase at 37°C for 3 h (McrBC) or 6 h (FspEI and MspJI), followed by analysis by electrophoresis on a 1% agarose gel with ethidium bromide.

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes
    Article Snippet: MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion. .. Then, 10 µl ligation master mix containing 0.2 µM each of two adaptors, 1 mM ATP and 800 U of T4 DNA ligase (NEB) was added to the digestion solution, and incubated for 6–12 h at 4°C.

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Polymerase Chain Reaction:

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: Paragraph title: PCR following treatment with a methylation-dependent restriction enzyme (PTMR) ... In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence .

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions. .. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and were subjected to single end sequencing on an Illumina HiSeq2500 sequencer.

    Infection:

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: .. One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture. ..

    Modification:

    Article Title: Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic)
    Article Snippet: Briefly, the total DNAs were digested by using the methylation-dependent restriction enzyme FspEI (NEB, USA). .. FspEI can recognize 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the Cm CGG and m CHG sites, and generate a double-stranded DNA break on the 30 side of the modified cytosine at a fixed distance (N12 /N16 ).

    Real-time Polymerase Chain Reaction:

    Article Title: Heritable Epigenetic Variation among Maize Inbreds
    Article Snippet: Paragraph title: qPCR ... 1 microgram of genomic DNA was digested for 16 hr with MspJI or FspEI (New England Biolabs).

    Purification:

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation
    Article Snippet: Equal amounts of genomic DNA (500 ng) were undigested or digested with the restriction enzymes HpaII, FspEI, or McrBC following the supplier's instructions (New England Biolabs). .. The resulting DNA samples were purified, further digested with Tru9I, resolved on an agarose gel, transferred to a Hybond-N+ membrane, and hybridized with a telomeric probe, as previously described ( ).

    Article Title: Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.)
    Article Snippet: The MethylRAD library was prepared by digesting genomic DNA using FspEI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 4 h. Then, the ligated products were amplified in 20 μl reactions. .. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and were subjected to single end sequencing on an Illumina HiSeq2500 sequencer.

    other:

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI
    Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

    Derivative Assay:

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture. .. Samples were digested for 2 h at 37°C and then heat inactivated at 80°C for 15 min. For coverage analysis, rarefaction analysis was performed on Illumina MiSeq sequencing reads derived from samples digested with MspJI or FspEI (digest and SWGA) or mock digested with no enzyme prior to SWGA with primer set pvset1920.

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  • 95
    New England Biolabs fspei
    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with <t>LpnPI,</t> followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and <t>FspEI</t> CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).
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    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Journal: Genome Research

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

    doi: 10.1101/gr.222885.117

    Figure Lengend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

    Techniques: Ligation, Fractionation, Amplification, Sequencing

    Schematic overview of the procedure for MethylRAD library preparation. Genomic DNA is digested with the restriction enzyme FspEI, producing 32-bp fragments including four-base 3′ overhangs. Adaptors with compatible overhangs (NNNN) are ligated to each end of these fragments. Tag density can be adjusted using adaptors with selective overhangs (e.g. NNNG). The constructs are amplified and purified by gel extraction. Sample-specific barcodes are incorporated in each construct by PCR, and the products pooled for sequencing.

    Journal: Open Biology

    Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

    doi: 10.1098/rsob.150130

    Figure Lengend Snippet: Schematic overview of the procedure for MethylRAD library preparation. Genomic DNA is digested with the restriction enzyme FspEI, producing 32-bp fragments including four-base 3′ overhangs. Adaptors with compatible overhangs (NNNN) are ligated to each end of these fragments. Tag density can be adjusted using adaptors with selective overhangs (e.g. NNNG). The constructs are amplified and purified by gel extraction. Sample-specific barcodes are incorporated in each construct by PCR, and the products pooled for sequencing.

    Article Snippet: MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion.

    Techniques: Construct, Amplification, Purification, Gel Extraction, Polymerase Chain Reaction, Sequencing

    Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: Sequencing, Construct

    Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.

    Journal: Genome Research

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation

    doi: 10.1101/gr.202465.115

    Figure Lengend Snippet: Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.

    Article Snippet: Equal amounts of genomic DNA (500 ng) were undigested or digested with the restriction enzymes HpaII, FspEI, or McrBC following the supplier's instructions (New England Biolabs).

    Techniques: Methylation, DNA Methylation Assay, Sequencing, Southern Blot, Hybridization, Staining