Structured Review

Macrogen amplicons
Induction of HCV escape from a direct acting antiviral in HFBR. ( a ) Timeline of Huh7.5 cell cultivation in DMEM + 10%FBS and serum-free AEM, indicating infection with HCV and initiation of treatment with different concentrations of the NS5A inhibitor daclatasvir (DCV). HCV from HFBR harvests (1000 μL, 50 μL or 5 μL) were used for infection of 10 6 Huh7.5 cells plated the previous day in T25 cell culture flasks (derived cultures). Peak infectivity titers in derived cultures are reported. Amino acid at NS5A position 93 according to the H77 reference sequence (GenBank accession no AF009606) was determined by Sanger sequencing for selected harvests and derived cultures. *, H at position 93 was present in ~50% of viral genomes. ( b ) 8 × 10 7 Huh7.5 cells were seeded in a hollow fiber bioreactor in DMEM + 10% FBS and infected with 1.25 × 10 6 FFU of HCV third passage stock on day 5 post cell seeding, when glucose consumption was 590 mg/day (arrow). At day 9 post cell seeding DMEM was replaced with serum-free AEM. Treatment with daclatasvir at 7.8 nM (corresponding to 64 x EC50 29 ) was initiated at day 27 post cell seeding; at day 45 the concentration was increased to 124.2 nM (corresponding to 1024 x EC50). On day 59 the treatment was terminated. ( c and d ) Determination of HCV infectivity titers and HCV RNA titers were carried out as described in Fig. 2 . *, indicates HCV infectivity titers below assay detection level. ( e ) HCV from harvest 9, 12 and 16 were loaded on 10–40% iodixanol gradients. Following ultracentrifugation 18 fractions were collected as in Fig. 5 . HCV infectivity and HCV RNA titers of each fraction were determined as described for Fig. 2 . ( f ) RNA extracted from harvest 9, 12 and 14 at 1-, 10-, 100- and 1000-fold dilution was used for RT-PCR for generation of a full-length amplicon spanning the complete HCV ORF. PCR products were visualized on a 1% agarose gel including a 1 kb DNA ladder; expected positions of full-length <t>amplicons</t> are indicated by arrows. Gel image was cropped as indicated by boxes; full-length gel is presented in Supplementary Fig. S4 .
Amplicons, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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92/100 stars

Images

1) Product Images from "High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals"

Article Title: High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

Journal: Scientific Reports

doi: 10.1038/s41598-018-35010-5

Induction of HCV escape from a direct acting antiviral in HFBR. ( a ) Timeline of Huh7.5 cell cultivation in DMEM + 10%FBS and serum-free AEM, indicating infection with HCV and initiation of treatment with different concentrations of the NS5A inhibitor daclatasvir (DCV). HCV from HFBR harvests (1000 μL, 50 μL or 5 μL) were used for infection of 10 6 Huh7.5 cells plated the previous day in T25 cell culture flasks (derived cultures). Peak infectivity titers in derived cultures are reported. Amino acid at NS5A position 93 according to the H77 reference sequence (GenBank accession no AF009606) was determined by Sanger sequencing for selected harvests and derived cultures. *, H at position 93 was present in ~50% of viral genomes. ( b ) 8 × 10 7 Huh7.5 cells were seeded in a hollow fiber bioreactor in DMEM + 10% FBS and infected with 1.25 × 10 6 FFU of HCV third passage stock on day 5 post cell seeding, when glucose consumption was 590 mg/day (arrow). At day 9 post cell seeding DMEM was replaced with serum-free AEM. Treatment with daclatasvir at 7.8 nM (corresponding to 64 x EC50 29 ) was initiated at day 27 post cell seeding; at day 45 the concentration was increased to 124.2 nM (corresponding to 1024 x EC50). On day 59 the treatment was terminated. ( c and d ) Determination of HCV infectivity titers and HCV RNA titers were carried out as described in Fig. 2 . *, indicates HCV infectivity titers below assay detection level. ( e ) HCV from harvest 9, 12 and 16 were loaded on 10–40% iodixanol gradients. Following ultracentrifugation 18 fractions were collected as in Fig. 5 . HCV infectivity and HCV RNA titers of each fraction were determined as described for Fig. 2 . ( f ) RNA extracted from harvest 9, 12 and 14 at 1-, 10-, 100- and 1000-fold dilution was used for RT-PCR for generation of a full-length amplicon spanning the complete HCV ORF. PCR products were visualized on a 1% agarose gel including a 1 kb DNA ladder; expected positions of full-length amplicons are indicated by arrows. Gel image was cropped as indicated by boxes; full-length gel is presented in Supplementary Fig. S4 .
Figure Legend Snippet: Induction of HCV escape from a direct acting antiviral in HFBR. ( a ) Timeline of Huh7.5 cell cultivation in DMEM + 10%FBS and serum-free AEM, indicating infection with HCV and initiation of treatment with different concentrations of the NS5A inhibitor daclatasvir (DCV). HCV from HFBR harvests (1000 μL, 50 μL or 5 μL) were used for infection of 10 6 Huh7.5 cells plated the previous day in T25 cell culture flasks (derived cultures). Peak infectivity titers in derived cultures are reported. Amino acid at NS5A position 93 according to the H77 reference sequence (GenBank accession no AF009606) was determined by Sanger sequencing for selected harvests and derived cultures. *, H at position 93 was present in ~50% of viral genomes. ( b ) 8 × 10 7 Huh7.5 cells were seeded in a hollow fiber bioreactor in DMEM + 10% FBS and infected with 1.25 × 10 6 FFU of HCV third passage stock on day 5 post cell seeding, when glucose consumption was 590 mg/day (arrow). At day 9 post cell seeding DMEM was replaced with serum-free AEM. Treatment with daclatasvir at 7.8 nM (corresponding to 64 x EC50 29 ) was initiated at day 27 post cell seeding; at day 45 the concentration was increased to 124.2 nM (corresponding to 1024 x EC50). On day 59 the treatment was terminated. ( c and d ) Determination of HCV infectivity titers and HCV RNA titers were carried out as described in Fig. 2 . *, indicates HCV infectivity titers below assay detection level. ( e ) HCV from harvest 9, 12 and 16 were loaded on 10–40% iodixanol gradients. Following ultracentrifugation 18 fractions were collected as in Fig. 5 . HCV infectivity and HCV RNA titers of each fraction were determined as described for Fig. 2 . ( f ) RNA extracted from harvest 9, 12 and 14 at 1-, 10-, 100- and 1000-fold dilution was used for RT-PCR for generation of a full-length amplicon spanning the complete HCV ORF. PCR products were visualized on a 1% agarose gel including a 1 kb DNA ladder; expected positions of full-length amplicons are indicated by arrows. Gel image was cropped as indicated by boxes; full-length gel is presented in Supplementary Fig. S4 .

Techniques Used: Infection, Cell Culture, Derivative Assay, Sequencing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

2) Product Images from "Novel Mutations in Sandhoff Disease: A Molecular Analysis among Iranian Cohort of Infantile Patients"

Article Title: Novel Mutations in Sandhoff Disease: A Molecular Analysis among Iranian Cohort of Infantile Patients

Journal: Iranian Journal of Public Health

doi:

Amplicons electrophoretic results of Exon 1–5 (exon 1, 2 and 3 in separate tubes and exon 4, 5 in same tube). Here only exon 4 and 5 depicted. M) marker 50bp, 1, 2) two normal samples with both exon 4, 5 HEXB and internal control primers, 3) normal sample with primers for exon 4, 5, 4) proband’s PCR product with both inernal and exon 4,5 primers, 5) proband’s PCR product with only exon 4, 5 primers
Figure Legend Snippet: Amplicons electrophoretic results of Exon 1–5 (exon 1, 2 and 3 in separate tubes and exon 4, 5 in same tube). Here only exon 4 and 5 depicted. M) marker 50bp, 1, 2) two normal samples with both exon 4, 5 HEXB and internal control primers, 3) normal sample with primers for exon 4, 5, 4) proband’s PCR product with both inernal and exon 4,5 primers, 5) proband’s PCR product with only exon 4, 5 primers

Techniques Used: Marker, Polymerase Chain Reaction

3) Product Images from "Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria"

Article Title: Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria

Journal: PLoS ONE

doi: 10.1371/journal.pone.0047071

SSCP results showing a migration pattern alteration in the exon 7 amplicons caused by the G829A substitution (10% acrylamide-bisacrylamide gel) - samples at the extremes (wild type isolate in the middle).
Figure Legend Snippet: SSCP results showing a migration pattern alteration in the exon 7 amplicons caused by the G829A substitution (10% acrylamide-bisacrylamide gel) - samples at the extremes (wild type isolate in the middle).

Techniques Used: Migration

Related Articles

Sequencing:

Article Title: Systematics of Huicundomantis, a new subgenus of Pristimantis ( Anura, Strabomantidae) with extraordinary cryptic diversity and eleven new species
Article Snippet: .. Amplicons were sequenced by the Macrogen Sequencing Team (Macrogen Inc., Seoul, Korea). .. The phylogenetic analyses were based on a 3199 bp dataset containing DNA sequences of the mitochondrial genes 16S (1318 bp, partial sequence), tRNALeu (90 bp), NADH dehydrogenase subunit 1 ND1 (961 bp), tRNAIle (73 bp), tRNAGln (71 bp), and tRNAMet (31 bp), and the nuclear gene RAG1 (655 bp).

Article Title: Molecular phylogeny and evolutionary history of Moricandia DC (Brassicaceae)
Article Snippet: .. Amplicons were sent to Macrogen Europe (Geumchun-gu, Seoul, Korea) for sequencing in both directions, using their corresponding PCR primers. .. Chromatograms were reviewed and contigs were produced using Geneious v. 9 ( ; Biomatters, Inc., San Francisco, CA, USA, http://www.geneious.com ) and thorough revised and corrected by eye inspection.

Article Title: High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals
Article Snippet: .. Obtained amplicons were subjected to Sanger sequencing (Macrogen Europe). ..

Article Title: Non-polio enteroviruses in faeces of children diagnosed with acute flaccid paralysis in Nigeria
Article Snippet: .. Amplicons were shipped to Macrogen Inc., Seoul, South Korea, for purification and sequencing. ..

Polymerase Chain Reaction:

Article Title: Molecular phylogeny and evolutionary history of Moricandia DC (Brassicaceae)
Article Snippet: .. Amplicons were sent to Macrogen Europe (Geumchun-gu, Seoul, Korea) for sequencing in both directions, using their corresponding PCR primers. .. Chromatograms were reviewed and contigs were produced using Geneious v. 9 ( ; Biomatters, Inc., San Francisco, CA, USA, http://www.geneious.com ) and thorough revised and corrected by eye inspection.

Article Title: Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria
Article Snippet: .. The exon 7, in which a mutation was identified, was then amplified in all samples from all groups by PCR with the specific primers and conditions indicated in and the amplicons were sequenced (Macrogen Inc., Korea). .. Statistical analysis The association between alleles and malaria outcome groups was assessed by Pearson's chi-square tests and Fisher's exact test, this latter considered when there were a few cases in each comparison group (less than five), using the Simple Interactive Statistical Analysis software (SISA) .

Amplification:

Article Title: Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria
Article Snippet: .. The exon 7, in which a mutation was identified, was then amplified in all samples from all groups by PCR with the specific primers and conditions indicated in and the amplicons were sequenced (Macrogen Inc., Korea). .. Statistical analysis The association between alleles and malaria outcome groups was assessed by Pearson's chi-square tests and Fisher's exact test, this latter considered when there were a few cases in each comparison group (less than five), using the Simple Interactive Statistical Analysis software (SISA) .

Mutagenesis:

Article Title: Novel Mutations in Sandhoff Disease: A Molecular Analysis among Iranian Cohort of Infantile Patients
Article Snippet: .. Subsequently all amplicons were sequenced by a 3730XL ABI sequencer (Macrogen, Korea).whenever a mutation was detected in an index patient, the parents were also investigated to confirm the finding. ..

Article Title: Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria
Article Snippet: .. The exon 7, in which a mutation was identified, was then amplified in all samples from all groups by PCR with the specific primers and conditions indicated in and the amplicons were sequenced (Macrogen Inc., Korea). .. Statistical analysis The association between alleles and malaria outcome groups was assessed by Pearson's chi-square tests and Fisher's exact test, this latter considered when there were a few cases in each comparison group (less than five), using the Simple Interactive Statistical Analysis software (SISA) .

Purification:

Article Title: Genetic, Cytogenetic and Morphological Trends in the Evolution of the Rhodnius (Triatominae: Rhodniini) Trans-Andean Group
Article Snippet: .. Amplicons were sent to Macrogen Inc., Korea to be purified and sequenced in both directions. .. Nucleotide Sequence Alignment and Diversity All sequences used here are available in GenBank (accession numbers KC543506–66, and GQ850481, FJ229357–60, JQ686674–89 for those reported in Gómez-Palacio et al., (2012)).

Article Title: Non-polio enteroviruses in faeces of children diagnosed with acute flaccid paralysis in Nigeria
Article Snippet: .. Amplicons were shipped to Macrogen Inc., Seoul, South Korea, for purification and sequencing. ..

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  • 92
    Macrogen amplicons
    Induction of HCV escape from a direct acting antiviral in HFBR. ( a ) Timeline of Huh7.5 cell cultivation in DMEM + 10%FBS and serum-free AEM, indicating infection with HCV and initiation of treatment with different concentrations of the NS5A inhibitor daclatasvir (DCV). HCV from HFBR harvests (1000 μL, 50 μL or 5 μL) were used for infection of 10 6 Huh7.5 cells plated the previous day in T25 cell culture flasks (derived cultures). Peak infectivity titers in derived cultures are reported. Amino acid at NS5A position 93 according to the H77 reference sequence (GenBank accession no AF009606) was determined by Sanger sequencing for selected harvests and derived cultures. *, H at position 93 was present in ~50% of viral genomes. ( b ) 8 × 10 7 Huh7.5 cells were seeded in a hollow fiber bioreactor in DMEM + 10% FBS and infected with 1.25 × 10 6 FFU of HCV third passage stock on day 5 post cell seeding, when glucose consumption was 590 mg/day (arrow). At day 9 post cell seeding DMEM was replaced with serum-free AEM. Treatment with daclatasvir at 7.8 nM (corresponding to 64 x EC50 29 ) was initiated at day 27 post cell seeding; at day 45 the concentration was increased to 124.2 nM (corresponding to 1024 x EC50). On day 59 the treatment was terminated. ( c and d ) Determination of HCV infectivity titers and HCV RNA titers were carried out as described in Fig. 2 . *, indicates HCV infectivity titers below assay detection level. ( e ) HCV from harvest 9, 12 and 16 were loaded on 10–40% iodixanol gradients. Following ultracentrifugation 18 fractions were collected as in Fig. 5 . HCV infectivity and HCV RNA titers of each fraction were determined as described for Fig. 2 . ( f ) RNA extracted from harvest 9, 12 and 14 at 1-, 10-, 100- and 1000-fold dilution was used for RT-PCR for generation of a full-length amplicon spanning the complete HCV ORF. PCR products were visualized on a 1% agarose gel including a 1 kb DNA ladder; expected positions of full-length <t>amplicons</t> are indicated by arrows. Gel image was cropped as indicated by boxes; full-length gel is presented in Supplementary Fig. S4 .
    Amplicons, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplicons/product/Macrogen
    Average 92 stars, based on 322 article reviews
    Price from $9.99 to $1999.99
    amplicons - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    90
    Macrogen linton 16s pcr amplicons
    Five MPN cultures amplified by various qPCR assays for Campylobacter (de Boer <t>Lv1-16S,</t> circles; Van Dyke 16S, triangles) and Arcobacter butzleri ( hsp60 ; squares). The five wells were determined to contain Arcobacter cryaerophilus (A), A. butzleri and C. lari (B and C), A. butzleri (D), and A. butzleri with a C. jejuni spike (E). (F) The same samples were run with the Yamazaki multiplex <t>PCR</t> (containing the <t>Linton</t> 16S assay amplicon), with individual lanes 1 to 5 corresponding to panels A to E. Lane 6, Yamazaki multiplex positive control; lane 7, no-template control; lane 8, 100-bp ladder. The Van Dyke 16S assay was able to amplify Campylobacter in the presence of Arcobacter , while the de Boer Lv1-16S assay did not, due to a cross-reaction with Arcobacter . The Yamazaki multiplex PCR was unable to identify the two samples containing C. lari (B and C) but could identify C. jejuni from a matrix spike sample (E). ΔRn, normalized fluorescence minus the background fluorescence, where normalized fluorescence refers to the ratio of the probe fluorescence to the fluorescence of the passive reference dye (ROX).
    Linton 16s Pcr Amplicons, supplied by Macrogen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/linton 16s pcr amplicons/product/Macrogen
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    Macrogen amplicon library
    Bar chart of the bacterial diversity based on 16S rRNA gene <t>amplicon</t> analysis of samples from the Iztaccihuatl volcano. Each bar represents the phylum indicated by percent diversity from the three regions of the volcano that were sampled.
    Amplicon Library, supplied by Macrogen, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplicon library/product/Macrogen
    Average 93 stars, based on 1 article reviews
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    amplicon library - by Bioz Stars, 2020-07
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    Induction of HCV escape from a direct acting antiviral in HFBR. ( a ) Timeline of Huh7.5 cell cultivation in DMEM + 10%FBS and serum-free AEM, indicating infection with HCV and initiation of treatment with different concentrations of the NS5A inhibitor daclatasvir (DCV). HCV from HFBR harvests (1000 μL, 50 μL or 5 μL) were used for infection of 10 6 Huh7.5 cells plated the previous day in T25 cell culture flasks (derived cultures). Peak infectivity titers in derived cultures are reported. Amino acid at NS5A position 93 according to the H77 reference sequence (GenBank accession no AF009606) was determined by Sanger sequencing for selected harvests and derived cultures. *, H at position 93 was present in ~50% of viral genomes. ( b ) 8 × 10 7 Huh7.5 cells were seeded in a hollow fiber bioreactor in DMEM + 10% FBS and infected with 1.25 × 10 6 FFU of HCV third passage stock on day 5 post cell seeding, when glucose consumption was 590 mg/day (arrow). At day 9 post cell seeding DMEM was replaced with serum-free AEM. Treatment with daclatasvir at 7.8 nM (corresponding to 64 x EC50 29 ) was initiated at day 27 post cell seeding; at day 45 the concentration was increased to 124.2 nM (corresponding to 1024 x EC50). On day 59 the treatment was terminated. ( c and d ) Determination of HCV infectivity titers and HCV RNA titers were carried out as described in Fig. 2 . *, indicates HCV infectivity titers below assay detection level. ( e ) HCV from harvest 9, 12 and 16 were loaded on 10–40% iodixanol gradients. Following ultracentrifugation 18 fractions were collected as in Fig. 5 . HCV infectivity and HCV RNA titers of each fraction were determined as described for Fig. 2 . ( f ) RNA extracted from harvest 9, 12 and 14 at 1-, 10-, 100- and 1000-fold dilution was used for RT-PCR for generation of a full-length amplicon spanning the complete HCV ORF. PCR products were visualized on a 1% agarose gel including a 1 kb DNA ladder; expected positions of full-length amplicons are indicated by arrows. Gel image was cropped as indicated by boxes; full-length gel is presented in Supplementary Fig. S4 .

    Journal: Scientific Reports

    Article Title: High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

    doi: 10.1038/s41598-018-35010-5

    Figure Lengend Snippet: Induction of HCV escape from a direct acting antiviral in HFBR. ( a ) Timeline of Huh7.5 cell cultivation in DMEM + 10%FBS and serum-free AEM, indicating infection with HCV and initiation of treatment with different concentrations of the NS5A inhibitor daclatasvir (DCV). HCV from HFBR harvests (1000 μL, 50 μL or 5 μL) were used for infection of 10 6 Huh7.5 cells plated the previous day in T25 cell culture flasks (derived cultures). Peak infectivity titers in derived cultures are reported. Amino acid at NS5A position 93 according to the H77 reference sequence (GenBank accession no AF009606) was determined by Sanger sequencing for selected harvests and derived cultures. *, H at position 93 was present in ~50% of viral genomes. ( b ) 8 × 10 7 Huh7.5 cells were seeded in a hollow fiber bioreactor in DMEM + 10% FBS and infected with 1.25 × 10 6 FFU of HCV third passage stock on day 5 post cell seeding, when glucose consumption was 590 mg/day (arrow). At day 9 post cell seeding DMEM was replaced with serum-free AEM. Treatment with daclatasvir at 7.8 nM (corresponding to 64 x EC50 29 ) was initiated at day 27 post cell seeding; at day 45 the concentration was increased to 124.2 nM (corresponding to 1024 x EC50). On day 59 the treatment was terminated. ( c and d ) Determination of HCV infectivity titers and HCV RNA titers were carried out as described in Fig. 2 . *, indicates HCV infectivity titers below assay detection level. ( e ) HCV from harvest 9, 12 and 16 were loaded on 10–40% iodixanol gradients. Following ultracentrifugation 18 fractions were collected as in Fig. 5 . HCV infectivity and HCV RNA titers of each fraction were determined as described for Fig. 2 . ( f ) RNA extracted from harvest 9, 12 and 14 at 1-, 10-, 100- and 1000-fold dilution was used for RT-PCR for generation of a full-length amplicon spanning the complete HCV ORF. PCR products were visualized on a 1% agarose gel including a 1 kb DNA ladder; expected positions of full-length amplicons are indicated by arrows. Gel image was cropped as indicated by boxes; full-length gel is presented in Supplementary Fig. S4 .

    Article Snippet: Obtained amplicons were subjected to Sanger sequencing (Macrogen Europe).

    Techniques: Infection, Cell Culture, Derivative Assay, Sequencing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Sequencing analysis of the on-target site for each CRISPR gRNA. ( A ) On-target sites were amplified from the extracted DNA of CRISPR-transduced 293 T cells in order to create at least 20 plasmid clones for each gRNA construct, and amplicons were Sanger sequenced. Wild-type NL4-3 sequences are shown topmost with the PAM site ( NGG ) indicated in blue. Mutations are indicated in red and frameshift mutations are denoted with a blue asterisk (*). Cas9 cleavage sites are indicated in red vertical lines. Percentage numbers at the left side of each sequence denote the frequency of the corresponding mutation pattern found within all sequenced samples for each gRNA. ( B ) Mutation frequency and distribution of mutation types for each gRNA, cumulative for tat -CRISPR and rev -CRISPR, and overall CRISPR-transduced samples.

    Journal: Scientific Reports

    Article Title: CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures

    doi: 10.1038/s41598-018-26190-1

    Figure Lengend Snippet: Sequencing analysis of the on-target site for each CRISPR gRNA. ( A ) On-target sites were amplified from the extracted DNA of CRISPR-transduced 293 T cells in order to create at least 20 plasmid clones for each gRNA construct, and amplicons were Sanger sequenced. Wild-type NL4-3 sequences are shown topmost with the PAM site ( NGG ) indicated in blue. Mutations are indicated in red and frameshift mutations are denoted with a blue asterisk (*). Cas9 cleavage sites are indicated in red vertical lines. Percentage numbers at the left side of each sequence denote the frequency of the corresponding mutation pattern found within all sequenced samples for each gRNA. ( B ) Mutation frequency and distribution of mutation types for each gRNA, cumulative for tat -CRISPR and rev -CRISPR, and overall CRISPR-transduced samples.

    Article Snippet: On-target amplicons were sequenced (Macrogen Inc.) and analyzed using GENETYX software ver.

    Techniques: Sequencing, CRISPR, Amplification, Plasmid Preparation, Clone Assay, Construct, Mutagenesis

    Five MPN cultures amplified by various qPCR assays for Campylobacter (de Boer Lv1-16S, circles; Van Dyke 16S, triangles) and Arcobacter butzleri ( hsp60 ; squares). The five wells were determined to contain Arcobacter cryaerophilus (A), A. butzleri and C. lari (B and C), A. butzleri (D), and A. butzleri with a C. jejuni spike (E). (F) The same samples were run with the Yamazaki multiplex PCR (containing the Linton 16S assay amplicon), with individual lanes 1 to 5 corresponding to panels A to E. Lane 6, Yamazaki multiplex positive control; lane 7, no-template control; lane 8, 100-bp ladder. The Van Dyke 16S assay was able to amplify Campylobacter in the presence of Arcobacter , while the de Boer Lv1-16S assay did not, due to a cross-reaction with Arcobacter . The Yamazaki multiplex PCR was unable to identify the two samples containing C. lari (B and C) but could identify C. jejuni from a matrix spike sample (E). ΔRn, normalized fluorescence minus the background fluorescence, where normalized fluorescence refers to the ratio of the probe fluorescence to the fluorescence of the passive reference dye (ROX).

    Journal: Applied and Environmental Microbiology

    Article Title: Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

    doi: 10.1128/AEM.00077-16

    Figure Lengend Snippet: Five MPN cultures amplified by various qPCR assays for Campylobacter (de Boer Lv1-16S, circles; Van Dyke 16S, triangles) and Arcobacter butzleri ( hsp60 ; squares). The five wells were determined to contain Arcobacter cryaerophilus (A), A. butzleri and C. lari (B and C), A. butzleri (D), and A. butzleri with a C. jejuni spike (E). (F) The same samples were run with the Yamazaki multiplex PCR (containing the Linton 16S assay amplicon), with individual lanes 1 to 5 corresponding to panels A to E. Lane 6, Yamazaki multiplex positive control; lane 7, no-template control; lane 8, 100-bp ladder. The Van Dyke 16S assay was able to amplify Campylobacter in the presence of Arcobacter , while the de Boer Lv1-16S assay did not, due to a cross-reaction with Arcobacter . The Yamazaki multiplex PCR was unable to identify the two samples containing C. lari (B and C) but could identify C. jejuni from a matrix spike sample (E). ΔRn, normalized fluorescence minus the background fluorescence, where normalized fluorescence refers to the ratio of the probe fluorescence to the fluorescence of the passive reference dye (ROX).

    Article Snippet: As the Yamazaki multiplex PCR assay did not always identify a putative Campylobacter species, the Linton 16S PCR amplicons were also sequenced by Sanger method sequencing (Macrogen, Seoul, South Korea), and the resulting DNA sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis to confirm identity. qPCR-positive wells were plated on Bolton agar, and putative Campylobacter colonies were enriched in Bolton broth overnight at 37°C, followed by PCR (described below) to confirm identity.

    Techniques: Amplification, Real-time Polymerase Chain Reaction, Multiplex Assay, Polymerase Chain Reaction, Positive Control, Fluorescence

    Bar chart of the bacterial diversity based on 16S rRNA gene amplicon analysis of samples from the Iztaccihuatl volcano. Each bar represents the phylum indicated by percent diversity from the three regions of the volcano that were sampled.

    Journal: Microbiology Resource Announcements

    Article Title: Bacterial Diversity Based on a 16S rRNA Gene Amplicon Data Set from a High-Altitude Crater Lake and Glacial Samples of the Iztaccihuatl Volcanic Complex (Mexico)

    doi: 10.1128/MRA.01636-18

    Figure Lengend Snippet: Bar chart of the bacterial diversity based on 16S rRNA gene amplicon analysis of samples from the Iztaccihuatl volcano. Each bar represents the phylum indicated by percent diversity from the three regions of the volcano that were sampled.

    Article Snippet: The purification, quantification, and sequencing of the amplicon library was done at Macrogen, Inc. (Seoul, Republic of Korea) and accomplished via the Illumina platform with a MiSeq system.

    Techniques: Amplification