nspi  (New England Biolabs)


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    Name:
    NspI
    Description:
    NspI 1 250 units
    Catalog Number:
    r0602l
    Price:
    277
    Size:
    1 250 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs nspi
    NspI
    NspI 1 250 units
    https://www.bioz.com/result/nspi/product/New England Biolabs
    Average 90 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    nspi - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements"

    Article Title: Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements

    Journal: Nature genetics

    doi: 10.1038/ng.613

    Androgen stimulation induces AR/TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human PCa. a–b , Sites closest to TMPRSS2-ERG breakpoints from 8 PCa cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (p = 0.010 and 0.013 respectively) in LAPC4 cells. Labeled sites (e.g., T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. c , DHT and TOP2B dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). d , SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Fig. 8, 9 for overview of SLOT). e , ChIP enrichment of AR and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. f–g , 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± SE of two to three experiments.
    Figure Legend Snippet: Androgen stimulation induces AR/TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human PCa. a–b , Sites closest to TMPRSS2-ERG breakpoints from 8 PCa cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (p = 0.010 and 0.013 respectively) in LAPC4 cells. Labeled sites (e.g., T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. c , DHT and TOP2B dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). d , SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Fig. 8, 9 for overview of SLOT). e , ChIP enrichment of AR and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. f–g , 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± SE of two to three experiments.

    Techniques Used: Labeling, Activity Assay, Chromatin Immunoprecipitation, Inhibition

    2) Product Images from "tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci"

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx853

    Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.
    Figure Legend Snippet: Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Techniques Used: Ligation, Sequencing, Amplification, Polymerase Chain Reaction

    3) Product Images from "Copy Number Variation at the APOL1 Locus"

    Article Title: Copy Number Variation at the APOL1 Locus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125410

    Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the PCR product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with NspI. Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.
    Figure Legend Snippet: Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the PCR product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with NspI. Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.

    Techniques Used: Sequencing, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, TaqMan Copy Number Assay, Variant Assay

    Related Articles

    Amplification:

    Article Title: Novel copy number variants in children with autism and additional developmental anomalies
    Article Snippet: .. Briefly, the assay uses 250ng of genomic DNA digested with Nsp I or Sty I restriction enzyme (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Titanium Taq (Clonetech). .. PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clonetech) and a 90 µg aliquot was fragmented using DNaseI.

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After digestion, an adaptor was linked to the restricted fragments, the reaction was diluted 4 × and the fragments were amplified by PCR.

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci
    Article Snippet: tGBS procedure Approximately 120 ng of genomic DNA from each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA, USA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA, USA), No. R0636L] in NEB CutSmart Buffer 4 in a 30-μl volume at 37°C for 1.5 h following the manufacturer’s protocol. .. The pooled, purified digestion-ligation product was used as the template for a single selective PCR reaction using a selective primer (100 μM), an amplification primer (100 μM) and Phusion High-Fidelity PCR Master Mix with HF Buffer [New England Biolabs (Beverly, MA, USA), No. M0531L].

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA). .. This was used as a template in PCR amplification using Titanium Taq (Clontech, Mountainview, CA) and a single primer complementary to the adaptor sequence.

    Article Title: A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy
    Article Snippet: Amplification by PCR was performed on 40 ng of genomic DNA with Taq DNA polymerase (Roche) or Amplitaq (Life Technologies). .. Presence of the FITM2 c.4G > T transversion was determined in 137 ethnically matched healthy controls by restriction analysis of amplicons encompassing FITM2 exon 1 (primers as indicated in Table S1 ), which were purified as described above and digested with Nsp I (New England Biolabs) in accordance with the manufacturer's protocol.

    Article Title: Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1 2
    Article Snippet: Briefly, 250 ng of genomic DNA was digested with Nsp I and Sty I (New England Biolabs, Inc, Ipswich, MA), respectively, and then ligated to Nsp or Sty adaptors. .. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labeled with a biotinylated nucleotide, and hybridized to a human SNP 6.0 array (Affymetrix) at 49°C for 17 hours.

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors. .. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 °C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix).

    Article Title: Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease
    Article Snippet: .. Two aliquots of 250 ng DNA were digested with Sty I and Nsp I (New England Biolabs, Ipswich, MA), ligated with respective oligonucleotide adaptors (Affymetrix), and amplified by polymerase chain reaction (PCR) (Clontech Laboratories, Mountain View, CA). .. Forty-five microliters of the Agencourt AMPure-purified (Beckman Coulter, Brea, CA) DNA was fragmented and labeled (Affymetrix).

    Article Title: Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation
    Article Snippet: 2.4 Affymetrix genome-wide SNP analysis For genome-wide SNP determination, an Affymetrix Genome-wide Human SNP Array (Nsp/Sty Assay Kit 5.0/6.0; carrying 909,622 SNP markers) processing with sample preparation, digestion, ligation, amplification, labelling, hybridization, staining and scanning were conducted according to the manufacturer's instructions. .. For donor and recipient, 250 ng of double-stranded genomic DNA starting material were digested with Nsp I and Sty I (New England BioLabs GmbH, Frankfurt, Germany).

    Article Title: Copy Number Variations and Primary Open-Angle Glaucoma
    Article Snippet: .. Briefly, the assay uses 250 ng of genomic DNA digested with Nsp I and Sty I (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Taq (Titanium; Clontech, Palo Alto, CA). .. PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clontech), and a 90-μg aliquot was fragmented using DNase I.

    Article Title: X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3
    Article Snippet: The 25-μl amplification reaction contained ∼75 ng genomic DNA, 10 pmol each primer, 1× Bioline buffer (Bioline), 0.875 U Biolase DNA polymerase (Bioline), 200 μ M each dNTP, and 15% glycerol. .. Digestion reactions consisted of ∼500 ng of the DNA fragment discussed above, 5 U Nsp I (NE Biolabs), 2 μl 10× digestion buffer, and H2 O double-distilled to 20 μl, incubated for 3 h prior to heat denaturation at 65°C for 20 min.

    Filtration:

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA). .. PCR products were purified from excess primers and salts by column filtration and the eluted products were fragmented using DNase I.

    DNA Ligation:

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA. .. To ligate the splinkerette adaptor to the digested genomic DNA, 10 µl of ligation mix [1 µl splinkerette adaptor, 3 µl 10× DNA ligation buffer (New England Biolabs, Ipswich, MA), 1 µl 400 U/µl T4-Ligase (New England Biolabs), and 5 µl bidestilled water] were added to each well.

    Microarray:

    Article Title: Novel copy number variants in children with autism and additional developmental anomalies
    Article Snippet: Paragraph title: Affymetrix GeneChip® human mapping 250K microarray ... Briefly, the assay uses 250ng of genomic DNA digested with Nsp I or Sty I restriction enzyme (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Titanium Taq (Clonetech).

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: Paragraph title: Affymetrix GeneChip mapping 6.0 microarray ... In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix).

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: This microarray allows genotyping of approximately 1.8 million single nucleotide polymorphisms (SNPs) and CNAs permitting thus to detect abnormalities with a median intermarker distance of around 1 kb. .. Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA).

    Article Title: Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1 2
    Article Snippet: SNP array experiments were performed using the high-resolution Affymetrix GeneChip Human Mapping 6.0 microarray (SNP 6.0; Affymetrix, Inc, Santa Clara, CA) containing 906,600 probes for SNPs and 946,000 probes for CNVs, with a median physical inter-marker distance of 680 bp. .. Briefly, 250 ng of genomic DNA was digested with Nsp I and Sty I (New England Biolabs, Inc, Ipswich, MA), respectively, and then ligated to Nsp or Sty adaptors.

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors. .. Array experiments were performed using the high-resolution Affymetrix CytoScan HD microarray (Affymetrix, Inc, Santa Clara, CA) containing 2,696,550 markers of which 1,953,246 are non-polymorphic markers and 750,000 SNPs with over 99 % accuracy to detect accurate breakpoint estimation as well as loss of heterozygosity (LOH) determination.

    Article Title: Copy Number Variations and Primary Open-Angle Glaucoma
    Article Snippet: Paragraph title: Human-Mapping 250K Microarray ... Briefly, the assay uses 250 ng of genomic DNA digested with Nsp I and Sty I (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Taq (Titanium; Clontech, Palo Alto, CA).

    Incubation:

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA. .. Mixtures were incubated for 2.5 hours at 60 °C followed by an inactivation step for 20 minutes at 80 °C before cooling down to 4 °C.

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci
    Article Snippet: tGBS procedure Approximately 120 ng of genomic DNA from each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA, USA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA, USA), No. R0636L] in NEB CutSmart Buffer 4 in a 30-μl volume at 37°C for 1.5 h following the manufacturer’s protocol. .. The T4 DNA ligase was inactivated by incubation at 80°C for 20 min. All digestion-ligation products were pooled and 1 ml of pooled product was purified using the QiaQuick PCR purification kit [QIAGEN (Valencia, CA, USA), No. 28106].

    Article Title: X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3
    Article Snippet: .. Digestion reactions consisted of ∼500 ng of the DNA fragment discussed above, 5 U Nsp I (NE Biolabs), 2 μl 10× digestion buffer, and H2 O double-distilled to 20 μl, incubated for 3 h prior to heat denaturation at 65°C for 20 min. .. Resulting fragments were resolved on 2% agarose gels and were visualized under UV light after ethidium bromide staining.

    Genome Wide:

    Article Title: Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease
    Article Snippet: Genotyping and copy number variant (CNV) detection were performed in all of the patients using the Genome-Wide Human SNP Array 6.0 kit (Affymetrix, Santa Clara, CA). .. Two aliquots of 250 ng DNA were digested with Sty I and Nsp I (New England Biolabs, Ipswich, MA), ligated with respective oligonucleotide adaptors (Affymetrix), and amplified by polymerase chain reaction (PCR) (Clontech Laboratories, Mountain View, CA).

    Article Title: Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation
    Article Snippet: Paragraph title: Affymetrix genome-wide SNP analysis ... For donor and recipient, 250 ng of double-stranded genomic DNA starting material were digested with Nsp I and Sty I (New England BioLabs GmbH, Frankfurt, Germany).

    Article Title: Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals
    Article Snippet: For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples. .. After the reaction with restriction enzymes, we followed the manufacturer's instructions for the Affymetrix Genome-wide Human SNP array 6.0.

    Hybridization:

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: The membrane with fixed DNA was prehybridized for 4 hours on 65 °C in Church buffer (1% bovine serum albumin, 1 mmol/l EDTA, 0.5 M NaPO4 pH 7.2, 7% SDS, supplemented with 100 µg/ml salmon sperm DNA), followed by overnight hybridization with the radioactively labeled neo probe in Church buffer at 65 °C. .. Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA.

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After purification using Magnetic Beads (Agencourt Bioscience Corporation, Beverly, MA, USA), 90 μ g of PCR products was fragmented and end labeled using 30 U/ μ l of terminal deoxynucleotidyl transferase, and then hybridized for 16–18 h to the Affymetrix 6.0 chip at 49°C in a GeneChip Hybridization Oven 640 (Affymetrix).

    Article Title: Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1 2
    Article Snippet: Briefly, 250 ng of genomic DNA was digested with Nsp I and Sty I (New England Biolabs, Inc, Ipswich, MA), respectively, and then ligated to Nsp or Sty adaptors. .. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip Scanner 30007G (Affymetrix).

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors. .. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 °C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix).

    Article Title: Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation
    Article Snippet: 2.4 Affymetrix genome-wide SNP analysis For genome-wide SNP determination, an Affymetrix Genome-wide Human SNP Array (Nsp/Sty Assay Kit 5.0/6.0; carrying 909,622 SNP markers) processing with sample preparation, digestion, ligation, amplification, labelling, hybridization, staining and scanning were conducted according to the manufacturer's instructions. .. For donor and recipient, 250 ng of double-stranded genomic DNA starting material were digested with Nsp I and Sty I (New England BioLabs GmbH, Frankfurt, Germany).

    Positive Control:

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: One sample of pooled normal genomic DNA, provided by Affymetrix, was used as experimental positive control. .. 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors.

    Ligation:

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA. .. To ligate the splinkerette adaptor to the digested genomic DNA, 10 µl of ligation mix [1 µl splinkerette adaptor, 3 µl 10× DNA ligation buffer (New England Biolabs, Ipswich, MA), 1 µl 400 U/µl T4-Ligase (New England Biolabs), and 5 µl bidestilled water] were added to each well.

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci
    Article Snippet: tGBS procedure Approximately 120 ng of genomic DNA from each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA, USA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA, USA), No. R0636L] in NEB CutSmart Buffer 4 in a 30-μl volume at 37°C for 1.5 h following the manufacturer’s protocol. .. Unique, barcoded oligos (100 μM) and a universal single-strand oligo (100 μM) were added to each sample for ligation with T4 DNA ligase [New England Biolabs (Beverly, MA, USA), No. R0602L].

    Article Title: Genomic Methods for Clinical and Translational Pain Research
    Article Snippet: Paragraph title: 2.1.3. Nsp Restriction Enzyme Digestion, Ligation, and PCR ... Nsp I (10 U/µl), NE Buffer 2 (10×), and BSA (100×; 10 mg/ml) from NEB, stored at −20°C.

    Mapping Assay:

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: Genotyping of DNA samples was performed with the Affymetrix GeneChipR Human SNP Mapping 6.0 array according to the GeneChip Mapping Assay Manual (Affymetrix, Santa Clara, CA). .. Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA).

    Generated:

    Article Title: X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3
    Article Snippet: A 474-bp amplification fragment containing the WASP alternate promoter and untranslated first exon was generated with the primer pair WAS-F and WAS-R. .. Digestion reactions consisted of ∼500 ng of the DNA fragment discussed above, 5 U Nsp I (NE Biolabs), 2 μl 10× digestion buffer, and H2 O double-distilled to 20 μl, incubated for 3 h prior to heat denaturation at 65°C for 20 min.

    Polymerase Chain Reaction:

    Article Title: Novel copy number variants in children with autism and additional developmental anomalies
    Article Snippet: .. Briefly, the assay uses 250ng of genomic DNA digested with Nsp I or Sty I restriction enzyme (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Titanium Taq (Clonetech). .. PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clonetech) and a 90 µg aliquot was fragmented using DNaseI.

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA. .. Ligation mixtures were incubated overnight at 16 °C in a PCR thermocycler (PTC-100 or PTC-200, MJ Research).

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After digestion, an adaptor was linked to the restricted fragments, the reaction was diluted 4 × and the fragments were amplified by PCR.

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci
    Article Snippet: tGBS procedure Approximately 120 ng of genomic DNA from each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA, USA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA, USA), No. R0636L] in NEB CutSmart Buffer 4 in a 30-μl volume at 37°C for 1.5 h following the manufacturer’s protocol. .. The T4 DNA ligase was inactivated by incubation at 80°C for 20 min. All digestion-ligation products were pooled and 1 ml of pooled product was purified using the QiaQuick PCR purification kit [QIAGEN (Valencia, CA, USA), No. 28106].

    Article Title: Genomic Methods for Clinical and Translational Pain Research
    Article Snippet: Paragraph title: 2.1.3. Nsp Restriction Enzyme Digestion, Ligation, and PCR ... Nsp I (10 U/µl), NE Buffer 2 (10×), and BSA (100×; 10 mg/ml) from NEB, stored at −20°C.

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA). .. This was used as a template in PCR amplification using Titanium Taq (Clontech, Mountainview, CA) and a single primer complementary to the adaptor sequence.

    Article Title: A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy
    Article Snippet: PCR fragments were purified with NucleoFast 96 PCR plates (Clontech) in accordance with the manufacturer's protocol. .. Presence of the FITM2 c.4G > T transversion was determined in 137 ethnically matched healthy controls by restriction analysis of amplicons encompassing FITM2 exon 1 (primers as indicated in Table S1 ), which were purified as described above and digested with Nsp I (New England Biolabs) in accordance with the manufacturer's protocol.

    Article Title: Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease
    Article Snippet: .. Two aliquots of 250 ng DNA were digested with Sty I and Nsp I (New England Biolabs, Ipswich, MA), ligated with respective oligonucleotide adaptors (Affymetrix), and amplified by polymerase chain reaction (PCR) (Clontech Laboratories, Mountain View, CA). .. Forty-five microliters of the Agencourt AMPure-purified (Beckman Coulter, Brea, CA) DNA was fragmented and labeled (Affymetrix).

    Article Title: The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and p53 Networks
    Article Snippet: .. The PCR product was digested with 5 U Nsp I (New England Biolabs, Ipswich, MA), at 37°C for 4 h. Since Nsp I recognizes the polymorphic sequence, a G allele is demonstrated by the presence of two fragments 109 and 69 bp in a gel. ..

    Article Title: Copy Number Variations and Primary Open-Angle Glaucoma
    Article Snippet: .. Briefly, the assay uses 250 ng of genomic DNA digested with Nsp I and Sty I (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Taq (Titanium; Clontech, Palo Alto, CA). .. PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clontech), and a 90-μg aliquot was fragmented using DNase I.

    Article Title: Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals
    Article Snippet: For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples. .. For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples.

    Article Title: X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3
    Article Snippet: The thermal profile was 1 cycle at 94°C for 3 min; 31 cycles at 94°C for 30 s, 66°C for 55 s, and 72°C for 1 min; and a final extension cycle at 72°C for 5 min. PCR gel-extraction purifications were performed with QIAquick gel extraction–purification columns, according to the manufacturer's specifications (Qiagen), and were eluted into 40 μl of 10 mM Tris Cl, pH 8.5. .. Digestion reactions consisted of ∼500 ng of the DNA fragment discussed above, 5 U Nsp I (NE Biolabs), 2 μl 10× digestion buffer, and H2 O double-distilled to 20 μl, incubated for 3 h prior to heat denaturation at 65°C for 20 min.

    Magnetic Beads:

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After purification using Magnetic Beads (Agencourt Bioscience Corporation, Beverly, MA, USA), 90 μ g of PCR products was fragmented and end labeled using 30 U/ μ l of terminal deoxynucleotidyl transferase, and then hybridized for 16–18 h to the Affymetrix 6.0 chip at 49°C in a GeneChip Hybridization Oven 640 (Affymetrix).

    Article Title: Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals
    Article Snippet: For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples. .. For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples.

    Labeling:

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: The membrane with fixed DNA was prehybridized for 4 hours on 65 °C in Church buffer (1% bovine serum albumin, 1 mmol/l EDTA, 0.5 M NaPO4 pH 7.2, 7% SDS, supplemented with 100 µg/ml salmon sperm DNA), followed by overnight hybridization with the radioactively labeled neo probe in Church buffer at 65 °C. .. Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA.

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After purification using Magnetic Beads (Agencourt Bioscience Corporation, Beverly, MA, USA), 90 μ g of PCR products was fragmented and end labeled using 30 U/ μ l of terminal deoxynucleotidyl transferase, and then hybridized for 16–18 h to the Affymetrix 6.0 chip at 49°C in a GeneChip Hybridization Oven 640 (Affymetrix).

    Article Title: Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1 2
    Article Snippet: Briefly, 250 ng of genomic DNA was digested with Nsp I and Sty I (New England Biolabs, Inc, Ipswich, MA), respectively, and then ligated to Nsp or Sty adaptors. .. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labeled with a biotinylated nucleotide, and hybridized to a human SNP 6.0 array (Affymetrix) at 49°C for 17 hours.

    Article Title: Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease
    Article Snippet: Two aliquots of 250 ng DNA were digested with Sty I and Nsp I (New England Biolabs, Ipswich, MA), ligated with respective oligonucleotide adaptors (Affymetrix), and amplified by polymerase chain reaction (PCR) (Clontech Laboratories, Mountain View, CA). .. Forty-five microliters of the Agencourt AMPure-purified (Beckman Coulter, Brea, CA) DNA was fragmented and labeled (Affymetrix).

    Purification:

    Article Title: Novel copy number variants in children with autism and additional developmental anomalies
    Article Snippet: Briefly, the assay uses 250ng of genomic DNA digested with Nsp I or Sty I restriction enzyme (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Titanium Taq (Clonetech). .. PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clonetech) and a 90 µg aliquot was fragmented using DNaseI.

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After purification using Magnetic Beads (Agencourt Bioscience Corporation, Beverly, MA, USA), 90 μ g of PCR products was fragmented and end labeled using 30 U/ μ l of terminal deoxynucleotidyl transferase, and then hybridized for 16–18 h to the Affymetrix 6.0 chip at 49°C in a GeneChip Hybridization Oven 640 (Affymetrix).

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci
    Article Snippet: tGBS procedure Approximately 120 ng of genomic DNA from each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA, USA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA, USA), No. R0636L] in NEB CutSmart Buffer 4 in a 30-μl volume at 37°C for 1.5 h following the manufacturer’s protocol. .. The T4 DNA ligase was inactivated by incubation at 80°C for 20 min. All digestion-ligation products were pooled and 1 ml of pooled product was purified using the QiaQuick PCR purification kit [QIAGEN (Valencia, CA, USA), No. 28106].

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA). .. PCR products were purified from excess primers and salts by column filtration and the eluted products were fragmented using DNase I.

    Article Title: A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy
    Article Snippet: .. Presence of the FITM2 c.4G > T transversion was determined in 137 ethnically matched healthy controls by restriction analysis of amplicons encompassing FITM2 exon 1 (primers as indicated in Table S1 ), which were purified as described above and digested with Nsp I (New England Biolabs) in accordance with the manufacturer's protocol. ..

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: CGH analysis DNA was purified from frozen tissues using DNeasy (Qiagen Inc., Valencia, CA). .. 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors.

    Article Title: Copy Number Variations and Primary Open-Angle Glaucoma
    Article Snippet: Briefly, the assay uses 250 ng of genomic DNA digested with Nsp I and Sty I (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Taq (Titanium; Clontech, Palo Alto, CA). .. PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clontech), and a 90-μg aliquot was fragmented using DNase I.

    Article Title: Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals
    Article Snippet: For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples. .. For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples.

    Sequencing:

    Article Title: Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics
    Article Snippet: Briefly, 250 ng of DNA was digested using either Sty I or Nsp I (New England Biolabs, Ipswich, MA). .. This was used as a template in PCR amplification using Titanium Taq (Clontech, Mountainview, CA) and a single primer complementary to the adaptor sequence.

    Article Title: A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy
    Article Snippet: Paragraph title: Sequence analysis; WES and Sanger sequencing ... Presence of the FITM2 c.4G > T transversion was determined in 137 ethnically matched healthy controls by restriction analysis of amplicons encompassing FITM2 exon 1 (primers as indicated in Table S1 ), which were purified as described above and digested with Nsp I (New England Biolabs) in accordance with the manufacturer's protocol.

    Article Title: The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and p53 Networks
    Article Snippet: .. The PCR product was digested with 5 U Nsp I (New England Biolabs, Ipswich, MA), at 37°C for 4 h. Since Nsp I recognizes the polymorphic sequence, a G allele is demonstrated by the presence of two fragments 109 and 69 bp in a gel. ..

    Staining:

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. The chips were washed, stained in GeneChip Fluidic Station 450 (Affymetrix) and scanned with Scanner 3000 7G (Affymetrix).

    Article Title: Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1Single Nucleotide Polymorphism Microarray Analysis in Cortisol-Secreting Adrenocortical Adenomas Identifies New Candidate Genes and Pathways 1 2
    Article Snippet: Briefly, 250 ng of genomic DNA was digested with Nsp I and Sty I (New England Biolabs, Inc, Ipswich, MA), respectively, and then ligated to Nsp or Sty adaptors. .. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip Scanner 30007G (Affymetrix).

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors. .. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 °C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix).

    Article Title: Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease
    Article Snippet: Two aliquots of 250 ng DNA were digested with Sty I and Nsp I (New England Biolabs, Ipswich, MA), ligated with respective oligonucleotide adaptors (Affymetrix), and amplified by polymerase chain reaction (PCR) (Clontech Laboratories, Mountain View, CA). .. The labeled DNAwas hybridized to the single nucleotide polymorphism (SNP) arrays for 16 hours, then washed, stained (Fluidics Station 450, Affymetrix), and scanned (Scanner, Affymetrix).

    Article Title: Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation
    Article Snippet: 2.4 Affymetrix genome-wide SNP analysis For genome-wide SNP determination, an Affymetrix Genome-wide Human SNP Array (Nsp/Sty Assay Kit 5.0/6.0; carrying 909,622 SNP markers) processing with sample preparation, digestion, ligation, amplification, labelling, hybridization, staining and scanning were conducted according to the manufacturer's instructions. .. For donor and recipient, 250 ng of double-stranded genomic DNA starting material were digested with Nsp I and Sty I (New England BioLabs GmbH, Frankfurt, Germany).

    Article Title: X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3
    Article Snippet: Digestion reactions consisted of ∼500 ng of the DNA fragment discussed above, 5 U Nsp I (NE Biolabs), 2 μl 10× digestion buffer, and H2 O double-distilled to 20 μl, incubated for 3 h prior to heat denaturation at 65°C for 20 min. .. Resulting fragments were resolved on 2% agarose gels and were visualized under UV light after ethidium bromide staining.

    Sample Prep:

    Article Title: Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation
    Article Snippet: 2.4 Affymetrix genome-wide SNP analysis For genome-wide SNP determination, an Affymetrix Genome-wide Human SNP Array (Nsp/Sty Assay Kit 5.0/6.0; carrying 909,622 SNP markers) processing with sample preparation, digestion, ligation, amplification, labelling, hybridization, staining and scanning were conducted according to the manufacturer's instructions. .. For donor and recipient, 250 ng of double-stranded genomic DNA starting material were digested with Nsp I and Sty I (New England BioLabs GmbH, Frankfurt, Germany).

    Chromatin Immunoprecipitation:

    Article Title: High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?
    Article Snippet: In two separate reactions, genomic DNA (250 ng) was digested with Nsp I or Sty I (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer (Affymetrix). .. After purification using Magnetic Beads (Agencourt Bioscience Corporation, Beverly, MA, USA), 90 μ g of PCR products was fragmented and end labeled using 30 U/ μ l of terminal deoxynucleotidyl transferase, and then hybridized for 16–18 h to the Affymetrix 6.0 chip at 49°C in a GeneChip Hybridization Oven 640 (Affymetrix).

    Article Title: Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis
    Article Snippet: 250 ng of genomic DNA was digested with Nsp I (New England Biolabs, Inc) and then ligated to Nsp adaptors. .. This chip covers 340 International Standards for Cytogenomic Arrays (ISCA) constitutional genes, 526 cancer genes (99.6 %) and 36,121 RefSeq genes.

    Software:

    Article Title: Comparative Analysis of Transposable Element Vector Systems in Human Cells
    Article Snippet: The densitometric evaluation of acquired radioactive signals was performed with the Aida 352 software, and a linear range of data obtained with reference samples was used to estimate the copy number in each sample. .. Genomic DNA was digested with Nsp I in 20 µl reactions per well consisting of 11 µl enzyme mix (10× NEB buffer, 10 U/µl enzyme, bidestilled water) and 9 µl genomic DNA.

    TaqMan SNP Genotyping Assay:

    Article Title: The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and p53 Networks
    Article Snippet: The PCR product was digested with 5 U Nsp I (New England Biolabs, Ipswich, MA), at 37°C for 4 h. Since Nsp I recognizes the polymorphic sequence, a G allele is demonstrated by the presence of two fragments 109 and 69 bp in a gel. .. The status of this SNP was determined also by using a Taqman SNP genotyping assay.

    Agarose Gel Electrophoresis:

    Article Title: Novel copy number variants in children with autism and additional developmental anomalies
    Article Snippet: Briefly, the assay uses 250ng of genomic DNA digested with Nsp I or Sty I restriction enzyme (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Titanium Taq (Clonetech). .. An aliquot of the fragmented DNA was separated and visualized in a 3% agarose gel in 1× TBE buffer to ensure that the bulk of the product had been properly fragmented.

    Article Title: Copy Number Variations and Primary Open-Angle Glaucoma
    Article Snippet: Briefly, the assay uses 250 ng of genomic DNA digested with Nsp I and Sty I (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Taq (Titanium; Clontech, Palo Alto, CA). .. An aliquot of the fragmented DNA was separated and visualized in a 3% agarose gel in 1× TBE buffer, to ensure that the bulk of the product had been properly fragmented.

    Spectrophotometry:

    Article Title: Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals
    Article Snippet: Genotyping 400 Japanese samples with SNP Array 6.0 platform The concentration of genomic DNA for all individuals was measured using a spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies). .. For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples.

    Concentration Assay:

    Article Title: Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals
    Article Snippet: .. For each individual assayed, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) by adding 6 μl for the 6 samples with low concentration (five samples for 1st set and one sample for 2nd set) and 5 μl for the remaining samples. ..

    Gel Extraction:

    Article Title: X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3
    Article Snippet: The thermal profile was 1 cycle at 94°C for 3 min; 31 cycles at 94°C for 30 s, 66°C for 55 s, and 72°C for 1 min; and a final extension cycle at 72°C for 5 min. PCR gel-extraction purifications were performed with QIAquick gel extraction–purification columns, according to the manufacturer's specifications (Qiagen), and were eluted into 40 μl of 10 mM Tris Cl, pH 8.5. .. Digestion reactions consisted of ∼500 ng of the DNA fragment discussed above, 5 U Nsp I (NE Biolabs), 2 μl 10× digestion buffer, and H2 O double-distilled to 20 μl, incubated for 3 h prior to heat denaturation at 65°C for 20 min.

    Variant Assay:

    Article Title: A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy
    Article Snippet: Presence of the FITM2 c.4G > T transversion was determined in 137 ethnically matched healthy controls by restriction analysis of amplicons encompassing FITM2 exon 1 (primers as indicated in Table S1 ), which were purified as described above and digested with Nsp I (New England Biolabs) in accordance with the manufacturer's protocol. .. The absence of the FITM2 c.4G > T variant was also verified in the Nijmegen WES database (5031 exomes) and ExAC (65,000 exomes).

    Article Title: Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease
    Article Snippet: Paragraph title: Array-Based Genotyping and Copy Number Variant Detection ... Two aliquots of 250 ng DNA were digested with Sty I and Nsp I (New England Biolabs, Ipswich, MA), ligated with respective oligonucleotide adaptors (Affymetrix), and amplified by polymerase chain reaction (PCR) (Clontech Laboratories, Mountain View, CA).

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    New England Biolabs r0602l
    R0602l, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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