pcr  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    NEB PCR Cloning Kit
    Description:
    NEB PCR Cloning Kit 20 rxns
    Catalog Number:
    E1202S
    Price:
    284
    Category:
    Cloning and Expression Systems
    Size:
    20 rxns
    Buy from Supplier


    Structured Review

    New England Biolabs pcr
    NEB PCR Cloning Kit
    NEB PCR Cloning Kit 20 rxns
    https://www.bioz.com/result/pcr/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr - by Bioz Stars, 2021-05
    99/100 stars

    Images

    Related Articles

    Clone Assay:

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: Each kmer was transcribed and purified according to the In Vitro Transcription of sgRNA protocol. .. Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2.0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F. .. The genomic target was amplified by PCR with Cloning Analysis Forward and Reverse Primers, mapping in the pMiniT 2.0 plasmid.

    Article Title: CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo
    Article Snippet: For deep sequencing analysis, new sets of primers were designed to permit the amplification of PCR products no longer than 350 bp. .. For the characterization of the nucleotide sequence of the hybrid exons, PCR products corresponding to the amplification of these hybrid exons were cloned into the pMiniT plasmid vector of a PCR cloning kit following the manufacturer’s instructions (NEB, Ipswich, MA). .. After transformation into bacteria and clone growth in a liquid medium, plasmidic DNA was extracted and purified for sequencing.

    Article Title: Single-stranded DNA and RNA origami
    Article Snippet: Phusion High-Fidelity PCR Master Mix with HF Buffer(100 reactions/50-liter volume) and Lambda Exonuclease (1000 units) was purchased from New England Biolabs, Inc. MinElute PCR Purification Kit was purchased from QIAGEN. .. Nicking endonuclease Nb.BbvCI (1000 units), restriction endonucleases Eco RI (5000 units), Xho I (5000 units) and Hind III (5000 units), T7 and T3 RNA polymerases (5000 units), PCR Cloning Kit (20 reactions), NEB 10-beta and NEB stable competent E. coli were purchased from New England Biolabs, Inc. pGEM-7zf (−) vector, Pure-yield plasmid miniprep system, and the Wizard SV Gel and PCR Clean-UP System were purchased from Promega. .. RNA Clean and Concentrator-25 was purchased from Zymo Research.

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: Each gRNA was transcribed and purified according to the In Vitro Transcription of sgRNA protocol. .. Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2 .0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F. .. The genomic target was amplified by PCR with Cloning Analysis Forward and Reverse Primers, mapping in the pMiniT 2 .0 plasmid.

    Plasmid Preparation:

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: Each kmer was transcribed and purified according to the In Vitro Transcription of sgRNA protocol. .. Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2.0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F. .. The genomic target was amplified by PCR with Cloning Analysis Forward and Reverse Primers, mapping in the pMiniT 2.0 plasmid.

    Article Title: CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo
    Article Snippet: For deep sequencing analysis, new sets of primers were designed to permit the amplification of PCR products no longer than 350 bp. .. For the characterization of the nucleotide sequence of the hybrid exons, PCR products corresponding to the amplification of these hybrid exons were cloned into the pMiniT plasmid vector of a PCR cloning kit following the manufacturer’s instructions (NEB, Ipswich, MA). .. After transformation into bacteria and clone growth in a liquid medium, plasmidic DNA was extracted and purified for sequencing.

    Article Title: Single-stranded DNA and RNA origami
    Article Snippet: Phusion High-Fidelity PCR Master Mix with HF Buffer(100 reactions/50-liter volume) and Lambda Exonuclease (1000 units) was purchased from New England Biolabs, Inc. MinElute PCR Purification Kit was purchased from QIAGEN. .. Nicking endonuclease Nb.BbvCI (1000 units), restriction endonucleases Eco RI (5000 units), Xho I (5000 units) and Hind III (5000 units), T7 and T3 RNA polymerases (5000 units), PCR Cloning Kit (20 reactions), NEB 10-beta and NEB stable competent E. coli were purchased from New England Biolabs, Inc. pGEM-7zf (−) vector, Pure-yield plasmid miniprep system, and the Wizard SV Gel and PCR Clean-UP System were purchased from Promega. .. RNA Clean and Concentrator-25 was purchased from Zymo Research.

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: Each gRNA was transcribed and purified according to the In Vitro Transcription of sgRNA protocol. .. Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2 .0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F. .. The genomic target was amplified by PCR with Cloning Analysis Forward and Reverse Primers, mapping in the pMiniT 2 .0 plasmid.

    Polymerase Chain Reaction:

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: Each kmer was transcribed and purified according to the In Vitro Transcription of sgRNA protocol. .. Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2.0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F. .. The genomic target was amplified by PCR with Cloning Analysis Forward and Reverse Primers, mapping in the pMiniT 2.0 plasmid.

    Article Title: CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo
    Article Snippet: For deep sequencing analysis, new sets of primers were designed to permit the amplification of PCR products no longer than 350 bp. .. For the characterization of the nucleotide sequence of the hybrid exons, PCR products corresponding to the amplification of these hybrid exons were cloned into the pMiniT plasmid vector of a PCR cloning kit following the manufacturer’s instructions (NEB, Ipswich, MA). .. After transformation into bacteria and clone growth in a liquid medium, plasmidic DNA was extracted and purified for sequencing.

    Article Title: Single-stranded DNA and RNA origami
    Article Snippet: Phusion High-Fidelity PCR Master Mix with HF Buffer(100 reactions/50-liter volume) and Lambda Exonuclease (1000 units) was purchased from New England Biolabs, Inc. MinElute PCR Purification Kit was purchased from QIAGEN. .. Nicking endonuclease Nb.BbvCI (1000 units), restriction endonucleases Eco RI (5000 units), Xho I (5000 units) and Hind III (5000 units), T7 and T3 RNA polymerases (5000 units), PCR Cloning Kit (20 reactions), NEB 10-beta and NEB stable competent E. coli were purchased from New England Biolabs, Inc. pGEM-7zf (−) vector, Pure-yield plasmid miniprep system, and the Wizard SV Gel and PCR Clean-UP System were purchased from Promega. .. RNA Clean and Concentrator-25 was purchased from Zymo Research.

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: Each gRNA was transcribed and purified according to the In Vitro Transcription of sgRNA protocol. .. Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2 .0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F. .. The genomic target was amplified by PCR with Cloning Analysis Forward and Reverse Primers, mapping in the pMiniT 2 .0 plasmid.

    Article Title: Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks
    Article Snippet: Enriched unmethylated DNA and input fractions were processed for microarray hybridization similar as described for the MIRA protocol . .. For genome amplification, DNA was incubated with T4 DNA polymerase (New England Biolabs) for 20 minutes at 12°C, purified with PCR purification kits and ligated with blunt end linker ( 5'-AGCAACTGTGCTATCCGAGGGAT and 5'-ATCCCTCGGA ) overnight at 16°C with T4 ligase (New England Biolabs). .. Genome amplification was performed in the exponential phase with Taq polymerase (Qiagen).

    Sequencing:

    Article Title: CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo
    Article Snippet: For deep sequencing analysis, new sets of primers were designed to permit the amplification of PCR products no longer than 350 bp. .. For the characterization of the nucleotide sequence of the hybrid exons, PCR products corresponding to the amplification of these hybrid exons were cloned into the pMiniT plasmid vector of a PCR cloning kit following the manufacturer’s instructions (NEB, Ipswich, MA). .. After transformation into bacteria and clone growth in a liquid medium, plasmidic DNA was extracted and purified for sequencing.

    Amplification:

    Article Title: CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo
    Article Snippet: For deep sequencing analysis, new sets of primers were designed to permit the amplification of PCR products no longer than 350 bp. .. For the characterization of the nucleotide sequence of the hybrid exons, PCR products corresponding to the amplification of these hybrid exons were cloned into the pMiniT plasmid vector of a PCR cloning kit following the manufacturer’s instructions (NEB, Ipswich, MA). .. After transformation into bacteria and clone growth in a liquid medium, plasmidic DNA was extracted and purified for sequencing.

    Article Title: Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks
    Article Snippet: Enriched unmethylated DNA and input fractions were processed for microarray hybridization similar as described for the MIRA protocol . .. For genome amplification, DNA was incubated with T4 DNA polymerase (New England Biolabs) for 20 minutes at 12°C, purified with PCR purification kits and ligated with blunt end linker ( 5'-AGCAACTGTGCTATCCGAGGGAT and 5'-ATCCCTCGGA ) overnight at 16°C with T4 ligase (New England Biolabs). .. Genome amplification was performed in the exponential phase with Taq polymerase (Qiagen).

    Incubation:

    Article Title: Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks
    Article Snippet: Enriched unmethylated DNA and input fractions were processed for microarray hybridization similar as described for the MIRA protocol . .. For genome amplification, DNA was incubated with T4 DNA polymerase (New England Biolabs) for 20 minutes at 12°C, purified with PCR purification kits and ligated with blunt end linker ( 5'-AGCAACTGTGCTATCCGAGGGAT and 5'-ATCCCTCGGA ) overnight at 16°C with T4 ligase (New England Biolabs). .. Genome amplification was performed in the exponential phase with Taq polymerase (Qiagen).

    Purification:

    Article Title: Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks
    Article Snippet: Enriched unmethylated DNA and input fractions were processed for microarray hybridization similar as described for the MIRA protocol . .. For genome amplification, DNA was incubated with T4 DNA polymerase (New England Biolabs) for 20 minutes at 12°C, purified with PCR purification kits and ligated with blunt end linker ( 5'-AGCAACTGTGCTATCCGAGGGAT and 5'-ATCCCTCGGA ) overnight at 16°C with T4 ligase (New England Biolabs). .. Genome amplification was performed in the exponential phase with Taq polymerase (Qiagen).

    Quantitative RT-PCR:

    Article Title: SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity
    Article Snippet: .. Next, we determined the limit of detection by comparing three different qRT-PCR kits obtained from New England Biolabs, Bio-Rad, and ThermoFisher Scientific ( ). .. As each kit specifies the use of slightly different PCR cycle times and temperatures, we first sought to standardize these into a “universal” thermocycler program to make it easier to switch between products when needed.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs pcr cloning kit
    Schematic of ssOrigami synthesis and replication by in vitro <t>PCR</t> and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind <t>III.</t> ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).
    Pcr Cloning Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr cloning kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr cloning kit - by Bioz Stars, 2021-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).

    Journal: Science (New York, N.Y.)

    Article Title: Single-stranded DNA and RNA origami

    doi: 10.1126/science.aao2648

    Figure Lengend Snippet: Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).

    Article Snippet: Nicking endonuclease Nb.BbvCI (1000 units), restriction endonucleases Eco RI (5000 units), Xho I (5000 units) and Hind III (5000 units), T7 and T3 RNA polymerases (5000 units), PCR Cloning Kit (20 reactions), NEB 10-beta and NEB stable competent E. coli were purchased from New England Biolabs, Inc. pGEM-7zf (−) vector, Pure-yield plasmid miniprep system, and the Wizard SV Gel and PCR Clean-UP System were purchased from Promega.

    Techniques: In Vitro, Polymerase Chain Reaction, In Vivo, Clone Assay, Sequencing, Modification, Ligation, Plasmid Preparation, Transformation Assay, Amplification, Produced

    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . The region of the Muc14a gene that was amplified contains at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9 / gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85D-cas9 transgene (1X), two copies of βtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85D-cas9/+ or nos-cas9/+ without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85D-cas9 males crossed to w females compared to w and βtub85D-cas9/+ control males crossed to w females. Left columns: embryos to pupae survival rate; central columns: pupae to adults survival rate and right columns: fraction of males and females in adults. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. **p

    Journal: PLoS Genetics

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    doi: 10.1371/journal.pgen.1008647

    Figure Lengend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . The region of the Muc14a gene that was amplified contains at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9 / gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85D-cas9 transgene (1X), two copies of βtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85D-cas9/+ or nos-cas9/+ without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85D-cas9 males crossed to w females compared to w and βtub85D-cas9/+ control males crossed to w females. Left columns: embryos to pupae survival rate; central columns: pupae to adults survival rate and right columns: fraction of males and females in adults. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. **p

    Article Snippet: Genomic DNA templates were obtained from Muc14a fragments previously cloned in a pMiniT 2 .0 vector using PCR Cloning Kit (NEB) and primers kmer Muc14_F and 58537687_F.

    Techniques: CRISPR, In Vitro, Amplification, Binding Assay, Polymerase Chain Reaction, Multiplex Assay

    Sensitivity of SalivaDirect Is Comparable to a Standard Approach for SARS-CoV-2 Detection in Saliva We compared Ct values for N1 between the modified CDC assay (nucleic acid extraction and singleplex qRT-PCR) and SalivaDirect for 41 saliva specimens tested with both methods. Overall, detection of SARS-CoV-2 with SalivaDirect is weaker (median 1.2 Ct, Wilcoxon; p

    Journal: Med (New York, N.y.)

    Article Title: SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity

    doi: 10.1016/j.medj.2020.12.010

    Figure Lengend Snippet: Sensitivity of SalivaDirect Is Comparable to a Standard Approach for SARS-CoV-2 Detection in Saliva We compared Ct values for N1 between the modified CDC assay (nucleic acid extraction and singleplex qRT-PCR) and SalivaDirect for 41 saliva specimens tested with both methods. Overall, detection of SARS-CoV-2 with SalivaDirect is weaker (median 1.2 Ct, Wilcoxon; p

    Article Snippet: Next, we determined the limit of detection by comparing three different qRT-PCR kits obtained from New England Biolabs, Bio-Rad, and ThermoFisher Scientific ( ).

    Techniques: Modification, CDC Assay, Quantitative RT-PCR

    SalivaDirect Is Highly Comparable to Standard qRT-PCR Tests with Nucleic Acid Extraction from Nasopharyngeal Swabs and Saliva We selected 37 paired positive and 30 paired negative nasopharyngeal swabs and saliva specimens. Paired samples were collected a maximum 4 days apart. Nasopharyngeal swabs and saliva specimens were tested with the ThermoFisher Scientific TaqPath COVID-19 combo kit, and average Ct values for N, S, and ORF1ab were compared to N1 Ct values for saliva specimens tested with SalivaDirect. (A) Comparison of 37 paired nasopharyngeal swabs and saliva tested with the TaqPath COVID-19 combo kit showed 84% positive agreement and no significant differences in each of the three virus targets (Wilcoxon; N: p = 0.51, S: p = 0.72, ORF1ab: p = 0.39). (B) Comparison of nasopharyngeal swabs tested with the TaqPatch COVID-19 combo kit and saliva tested with SalivaDirect showed 94% positive agreement. Median N1 Ct values were 3.3 Ct higher for SalivaDirect (Wilcoxon; p

    Journal: Med (New York, N.y.)

    Article Title: SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity

    doi: 10.1016/j.medj.2020.12.010

    Figure Lengend Snippet: SalivaDirect Is Highly Comparable to Standard qRT-PCR Tests with Nucleic Acid Extraction from Nasopharyngeal Swabs and Saliva We selected 37 paired positive and 30 paired negative nasopharyngeal swabs and saliva specimens. Paired samples were collected a maximum 4 days apart. Nasopharyngeal swabs and saliva specimens were tested with the ThermoFisher Scientific TaqPath COVID-19 combo kit, and average Ct values for N, S, and ORF1ab were compared to N1 Ct values for saliva specimens tested with SalivaDirect. (A) Comparison of 37 paired nasopharyngeal swabs and saliva tested with the TaqPath COVID-19 combo kit showed 84% positive agreement and no significant differences in each of the three virus targets (Wilcoxon; N: p = 0.51, S: p = 0.72, ORF1ab: p = 0.39). (B) Comparison of nasopharyngeal swabs tested with the TaqPatch COVID-19 combo kit and saliva tested with SalivaDirect showed 94% positive agreement. Median N1 Ct values were 3.3 Ct higher for SalivaDirect (Wilcoxon; p

    Article Snippet: Next, we determined the limit of detection by comparing three different qRT-PCR kits obtained from New England Biolabs, Bio-Rad, and ThermoFisher Scientific ( ).

    Techniques: Quantitative RT-PCR

    SalivaDirect Is a Simplified Method for SARS-CoV-2 Detection (A) Schematic overview of SalivaDirect workflow depicting the main steps of mixing saliva with proteinase K, heat inactivation, and dualplex qRT-PCR testing. Figure created with Biorender.com . (B) SARS-CoV-2 is stable in saliva for at least 7 days at 4°C, room temperature (RT; ∼19°C), and 30°C without addition of stabilizing buffers. Spiked-in saliva samples of low virus concentrations (12, 25, and 50 SARS-CoV-2 copies/μL) were kept at the indicated temperature for 7 days and then tested with SalivaDirect. N1 cycle threshold (Ct) values were lower when kept for 7 days at 30°C as compared to fresh specimens (Kruskal-Wallis; p = 0.03). Horizontal bars indicate the median. (C) Comparing Ct values for saliva treated with proteinase K and heat as compared to nucleic extraction yields higher N1 Ct values without extraction (Wilcoxon; p

    Journal: Med (New York, N.y.)

    Article Title: SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity

    doi: 10.1016/j.medj.2020.12.010

    Figure Lengend Snippet: SalivaDirect Is a Simplified Method for SARS-CoV-2 Detection (A) Schematic overview of SalivaDirect workflow depicting the main steps of mixing saliva with proteinase K, heat inactivation, and dualplex qRT-PCR testing. Figure created with Biorender.com . (B) SARS-CoV-2 is stable in saliva for at least 7 days at 4°C, room temperature (RT; ∼19°C), and 30°C without addition of stabilizing buffers. Spiked-in saliva samples of low virus concentrations (12, 25, and 50 SARS-CoV-2 copies/μL) were kept at the indicated temperature for 7 days and then tested with SalivaDirect. N1 cycle threshold (Ct) values were lower when kept for 7 days at 30°C as compared to fresh specimens (Kruskal-Wallis; p = 0.03). Horizontal bars indicate the median. (C) Comparing Ct values for saliva treated with proteinase K and heat as compared to nucleic extraction yields higher N1 Ct values without extraction (Wilcoxon; p

    Article Snippet: Next, we determined the limit of detection by comparing three different qRT-PCR kits obtained from New England Biolabs, Bio-Rad, and ThermoFisher Scientific ( ).

    Techniques: Quantitative RT-PCR

    SalivaDirect Is Validated for Use with Reagents and Instruments from Multiple Vendors We determined the lower limit of detection of SalivaDirect with a 2-fold dilution series (400, 200, 100, 50, 25, 12, and 6 copies/μL) of positive saliva spiked-in negative saliva. Initially, each concentration and negative saliva were tested in triplicate to determine the preliminary limit of detection (dark-colored dots). The limit of detection was confirmed with 20 additional replicates (light-colored dots) for which 19 out of 20 needed to be detected. Limit of detection when tested with (A–C) proteinase K, (D–F) RT-qPCR kits, and (G–I) qRT-PCR instruments from different vendors, while keeping the other conditions constant. (A) and (D), as well as (F) and (G), are duplicates to enable comparisons between the different combinations of reagents or instruments within a single row. Shown are the Ct values for the N1 primer-probe set. The horizontal bars indicate the median and the dotted line indicates the limit of detection. Data used to make this figure can be found in Data S1 .

    Journal: Med (New York, N.y.)

    Article Title: SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity

    doi: 10.1016/j.medj.2020.12.010

    Figure Lengend Snippet: SalivaDirect Is Validated for Use with Reagents and Instruments from Multiple Vendors We determined the lower limit of detection of SalivaDirect with a 2-fold dilution series (400, 200, 100, 50, 25, 12, and 6 copies/μL) of positive saliva spiked-in negative saliva. Initially, each concentration and negative saliva were tested in triplicate to determine the preliminary limit of detection (dark-colored dots). The limit of detection was confirmed with 20 additional replicates (light-colored dots) for which 19 out of 20 needed to be detected. Limit of detection when tested with (A–C) proteinase K, (D–F) RT-qPCR kits, and (G–I) qRT-PCR instruments from different vendors, while keeping the other conditions constant. (A) and (D), as well as (F) and (G), are duplicates to enable comparisons between the different combinations of reagents or instruments within a single row. Shown are the Ct values for the N1 primer-probe set. The horizontal bars indicate the median and the dotted line indicates the limit of detection. Data used to make this figure can be found in Data S1 .

    Article Snippet: Next, we determined the limit of detection by comparing three different qRT-PCR kits obtained from New England Biolabs, Bio-Rad, and ThermoFisher Scientific ( ).

    Techniques: Concentration Assay, Quantitative RT-PCR

    Epigenetic changes after SETD2 knockdown. A . Western blot for HBEC cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. B . Composite profile of epigenetic changes after SETD2 siRNA knockdown in HBEC. The profile was created for gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. C . Analysis of DNA methylation in the gene body of the NOTCH3 gene in conditions of H3K36me3 deficiency. The H3K36me3 profile of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfections in HBEC cells is shown. The region analyzed by COBRA methylation assays is indicated by a box. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with HpyCH4IV recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. D . Analysis of H3K36me3 and DNA methylation in the gene body of RFX2 in conditions of H3K36me3 deficiency. The region analyzed by bisulfite sequencing is marked by a box. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 20 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences. E . Western blot for HCT116-DKO cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. F . DNA methylation analysis of the gene body of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfection of HCT116-DKO cells. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 13 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences.

    Journal: PLoS ONE

    Article Title: Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks

    doi: 10.1371/journal.pone.0018844

    Figure Lengend Snippet: Epigenetic changes after SETD2 knockdown. A . Western blot for HBEC cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. B . Composite profile of epigenetic changes after SETD2 siRNA knockdown in HBEC. The profile was created for gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. C . Analysis of DNA methylation in the gene body of the NOTCH3 gene in conditions of H3K36me3 deficiency. The H3K36me3 profile of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfections in HBEC cells is shown. The region analyzed by COBRA methylation assays is indicated by a box. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with HpyCH4IV recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. D . Analysis of H3K36me3 and DNA methylation in the gene body of RFX2 in conditions of H3K36me3 deficiency. The region analyzed by bisulfite sequencing is marked by a box. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 20 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences. E . Western blot for HCT116-DKO cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. F . DNA methylation analysis of the gene body of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfection of HCT116-DKO cells. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 13 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences.

    Article Snippet: For genome amplification, DNA was incubated with T4 DNA polymerase (New England Biolabs) for 20 minutes at 12°C, purified with PCR purification kits and ligated with blunt end linker ( 5'-AGCAACTGTGCTATCCGAGGGAT and 5'-ATCCCTCGGA ) overnight at 16°C with T4 ligase (New England Biolabs).

    Techniques: Western Blot, Transfection, DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, Methylation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Vitro, Positive Control, Methylation Sequencing, Clone Assay