amplicons  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Thermo Fisher amplicons
    a Multiplex RT-PCR without internal control. Lanes 1 and 2 amplified product of M7 was loaded showing specific amplification of four all pathogens except ACLSV; lane 3 and 4 contains: product of RC and Gd-3; lane 5 and 8 contains amplicon of Gd-7; lane 6 and 13 100 bp marker (Genedirex); lane 7: product of sample 106; lanes 9 and 10 amplified product of Gd-1; lane 11 and 12 <t>amplicons</t> of T6/109 sample. b Multiplex RT-PCR with internal control ( nad5 ). Lanes 1–5: amplicons of samples RC, T9/109, Rd-1, Gd-6 and Gd-7; lanes 7–10 amplicons of Kw-2, Kw-3, K-4 and Kw-4; lane 6 100 bp marker. All products were fractionated on a 3 % agarose gel
    Amplicons, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2047 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplicons/product/Thermo Fisher
    Average 95 stars, based on 2047 article reviews
    Price from $9.99 to $1999.99
    amplicons - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "Simultaneous Detection of Major Pome Fruit Viruses and a Viroid"

    Article Title: Simultaneous Detection of Major Pome Fruit Viruses and a Viroid

    Journal: Indian Journal of Microbiology

    doi: 10.1007/s12088-013-0431-y

    a Multiplex RT-PCR without internal control. Lanes 1 and 2 amplified product of M7 was loaded showing specific amplification of four all pathogens except ACLSV; lane 3 and 4 contains: product of RC and Gd-3; lane 5 and 8 contains amplicon of Gd-7; lane 6 and 13 100 bp marker (Genedirex); lane 7: product of sample 106; lanes 9 and 10 amplified product of Gd-1; lane 11 and 12 amplicons of T6/109 sample. b Multiplex RT-PCR with internal control ( nad5 ). Lanes 1–5: amplicons of samples RC, T9/109, Rd-1, Gd-6 and Gd-7; lanes 7–10 amplicons of Kw-2, Kw-3, K-4 and Kw-4; lane 6 100 bp marker. All products were fractionated on a 3 % agarose gel
    Figure Legend Snippet: a Multiplex RT-PCR without internal control. Lanes 1 and 2 amplified product of M7 was loaded showing specific amplification of four all pathogens except ACLSV; lane 3 and 4 contains: product of RC and Gd-3; lane 5 and 8 contains amplicon of Gd-7; lane 6 and 13 100 bp marker (Genedirex); lane 7: product of sample 106; lanes 9 and 10 amplified product of Gd-1; lane 11 and 12 amplicons of T6/109 sample. b Multiplex RT-PCR with internal control ( nad5 ). Lanes 1–5: amplicons of samples RC, T9/109, Rd-1, Gd-6 and Gd-7; lanes 7–10 amplicons of Kw-2, Kw-3, K-4 and Kw-4; lane 6 100 bp marker. All products were fractionated on a 3 % agarose gel

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Agarose Gel Electrophoresis

    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start Taq DNA polymerase at same dilutions; lane M 100 bp marker
    Figure Legend Snippet: Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start Taq DNA polymerase at same dilutions; lane M 100 bp marker

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Multiplexing, Marker

    2) Product Images from "Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR(R)Green Real-Time PCR Methods and High Resolution Melting Analysis"

    Article Title: Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR(R)Green Real-Time PCR Methods and High Resolution Melting Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039287

    Melting peaks of amplicons in (A) triplex and (B) duplex PCR. Respective targets are indicated on each graph.
    Figure Legend Snippet: Melting peaks of amplicons in (A) triplex and (B) duplex PCR. Respective targets are indicated on each graph.

    Techniques Used: Polymerase Chain Reaction

    3) Product Images from "Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer"

    Article Title: Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer

    Journal: Scientific Reports

    doi: 10.1038/srep00602

    (A) Schematic representation of isoforms of Tctra pre-mRNA showing the primer positions and region used for the preparation of dsRNA.Boxes show exons and lines show introns and the numbers in the exons show sizes (bp). Common regions are shown in blue and male-specific regions are shown in red color. Green horizontal bar shows the region used to prepare Tctra dsRNA. Black dots represent the presence of stop codons in male-specific exons. Arrows represent the primer locations. Location of putative Tra/Tra2 binding sites are marked using green and yellow vertical lines. RBP1 binding sites are shown using red vertical lines. ISS sites are shown by black triangles. Primers shown as F and female-specific R were used for quantifying female-specific isoform. Primers shown as male-specific F and R2 were used for quantifying male-specific isoforms. (B) Gel picture showing the amplicons generated by PCR using sex-specific c-DNA as templates and F and R1 primers of Tctra .
    Figure Legend Snippet: (A) Schematic representation of isoforms of Tctra pre-mRNA showing the primer positions and region used for the preparation of dsRNA.Boxes show exons and lines show introns and the numbers in the exons show sizes (bp). Common regions are shown in blue and male-specific regions are shown in red color. Green horizontal bar shows the region used to prepare Tctra dsRNA. Black dots represent the presence of stop codons in male-specific exons. Arrows represent the primer locations. Location of putative Tra/Tra2 binding sites are marked using green and yellow vertical lines. RBP1 binding sites are shown using red vertical lines. ISS sites are shown by black triangles. Primers shown as F and female-specific R were used for quantifying female-specific isoform. Primers shown as male-specific F and R2 were used for quantifying male-specific isoforms. (B) Gel picture showing the amplicons generated by PCR using sex-specific c-DNA as templates and F and R1 primers of Tctra .

    Techniques Used: Binding Assay, Generated, Polymerase Chain Reaction

    4) Product Images from "Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius"

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    Journal: Bioscience Reports

    doi: 10.1042/BSR20140026

    Expression patterns of Ct PAL and Ct CHS genes after wounding (A) and during salinity stress (B) Samplings were carried out at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.
    Figure Legend Snippet: Expression patterns of Ct PAL and Ct CHS genes after wounding (A) and during salinity stress (B) Samplings were carried out at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.

    Techniques Used: Expressing, HAT Assay, Polymerase Chain Reaction

    Expression patterns of Ct PAL and Ct CHS genes after SA treatment with 0.1 mM (A) and 1 mM (B) concentrations Samplings were done at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.
    Figure Legend Snippet: Expression patterns of Ct PAL and Ct CHS genes after SA treatment with 0.1 mM (A) and 1 mM (B) concentrations Samplings were done at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.

    Techniques Used: Expressing, HAT Assay, Polymerase Chain Reaction

    5) Product Images from "Many LINE1 elements contribute to the transcriptome of human somatic cells"

    Article Title: Many LINE1 elements contribute to the transcriptome of human somatic cells

    Journal: Genome Biology

    doi: 10.1186/gb-2009-10-9-r100

    Characterization of L1 at 4p15.32. (a) Diagram of L1 at 4p15.32 and the surrounding region. The arrow designates the L1 transcript. Blue boxes indicate exons of the CD38 gene, with exon number designated. Oligonucleotides CD38-a and CD38-b are indicated. Unmarked triangles indicate the positions of oligonucleotides used in L1 TaqMan qPCR assay. (b) Alignments of L1 at 4p15.32 3' end and related sequences. 'chr 4 short tag' - the major 3' expression tag cloned from this site. 'chr4 long tag' - longer 3' expression tag and 3' RACE sequence cloned from this site. 'chr6 transduction' - paralogous, transduced sequence downstream of L1 on chromosome 6. 'U35' - similar distinct 3' expression tag that cannot be mapped to the human reference genome. 3' end target site duplications are highlighted in blue. Single nucleotide differences in the chromosome 6 sequence are highlighted in dark red. (c) Diagram of the pedigree of the CEPH/UTAH individuals used in this study. (d) Relative expression of the L1 at 4p15.32 in lymphoblastoid cell lines from CEPH individuals. Expression is in arbitrary units normalized to HPRT1. Error bars indicate ± standard deviation from three replicates. (e) Expression of CEPH individuals of the L1 at 4p15.32 compared to flanking exons of CD38 , normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. All data are representative of at least two biological replicates.
    Figure Legend Snippet: Characterization of L1 at 4p15.32. (a) Diagram of L1 at 4p15.32 and the surrounding region. The arrow designates the L1 transcript. Blue boxes indicate exons of the CD38 gene, with exon number designated. Oligonucleotides CD38-a and CD38-b are indicated. Unmarked triangles indicate the positions of oligonucleotides used in L1 TaqMan qPCR assay. (b) Alignments of L1 at 4p15.32 3' end and related sequences. 'chr 4 short tag' - the major 3' expression tag cloned from this site. 'chr4 long tag' - longer 3' expression tag and 3' RACE sequence cloned from this site. 'chr6 transduction' - paralogous, transduced sequence downstream of L1 on chromosome 6. 'U35' - similar distinct 3' expression tag that cannot be mapped to the human reference genome. 3' end target site duplications are highlighted in blue. Single nucleotide differences in the chromosome 6 sequence are highlighted in dark red. (c) Diagram of the pedigree of the CEPH/UTAH individuals used in this study. (d) Relative expression of the L1 at 4p15.32 in lymphoblastoid cell lines from CEPH individuals. Expression is in arbitrary units normalized to HPRT1. Error bars indicate ± standard deviation from three replicates. (e) Expression of CEPH individuals of the L1 at 4p15.32 compared to flanking exons of CD38 , normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. All data are representative of at least two biological replicates.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Clone Assay, Sequencing, Transduction, Standard Deviation

    Characterization of L1 at 13q14.2. (a) Diagram of L1 at 13q14.2 and the surrounding region. The arrow designates the L1 transcript. Triangles at F and R indicate positions of oligonucleotides 13q14.2F and 13q14.2R. Blue boxes indicate exons of the RB1 gene, with exon number designated. Oligonucleotides RB1-1 and RB1-2 are indicated. The sequence of the 3' expression tag is provided. (b) Relative expression of the L1 at 13q14.2 in lymphoblastoid cell lines from CEPH individuals. Expression is in arbitrary units normalized to HPRT1. Error bars indicate ± standard deviation from three replicates. (c) Expression of CEPH individuals of the L1 at 13q14.2 compared to flanking exons from RB1, normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. All data are representative of at least two biological replicates.
    Figure Legend Snippet: Characterization of L1 at 13q14.2. (a) Diagram of L1 at 13q14.2 and the surrounding region. The arrow designates the L1 transcript. Triangles at F and R indicate positions of oligonucleotides 13q14.2F and 13q14.2R. Blue boxes indicate exons of the RB1 gene, with exon number designated. Oligonucleotides RB1-1 and RB1-2 are indicated. The sequence of the 3' expression tag is provided. (b) Relative expression of the L1 at 13q14.2 in lymphoblastoid cell lines from CEPH individuals. Expression is in arbitrary units normalized to HPRT1. Error bars indicate ± standard deviation from three replicates. (c) Expression of CEPH individuals of the L1 at 13q14.2 compared to flanking exons from RB1, normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. All data are representative of at least two biological replicates.

    Techniques Used: Sequencing, Expressing, Standard Deviation

    Characterization of L1 at 6p22.2. (a) Diagram of L1 at 6p22.2 and the surrounding region. The arrow designates the L1 transcript. Triangles at F and R indicate positions of oligonucleotides 6p22F and 6p22R. Blue boxes indicate exons of the FAM65B gene, with exon number designated. Oligonucleotides FAM65B-1 and FAM65B-2 are indicated. The sequence of the 3' expression tag is provided. (b) Expression of CEPH individuals GM10861 and GM11994 of the L1 at 6P22.2 compared to flanking exons from FAM65B , normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. Data are representative of two experimental replicates.
    Figure Legend Snippet: Characterization of L1 at 6p22.2. (a) Diagram of L1 at 6p22.2 and the surrounding region. The arrow designates the L1 transcript. Triangles at F and R indicate positions of oligonucleotides 6p22F and 6p22R. Blue boxes indicate exons of the FAM65B gene, with exon number designated. Oligonucleotides FAM65B-1 and FAM65B-2 are indicated. The sequence of the 3' expression tag is provided. (b) Expression of CEPH individuals GM10861 and GM11994 of the L1 at 6P22.2 compared to flanking exons from FAM65B , normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. Data are representative of two experimental replicates.

    Techniques Used: Sequencing, Expressing

    6) Product Images from "TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)"

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1348-0

    PCR amplicons of AhLOX7 _5', AhLOX7_3 ', AhPLD1 , and AhPLD2. PCR amplification of AhLOX7_5 ', AhLOX7 _3', AhPLD1 , and AhPLD2 . a . Primers 2199/2184 amplify AhLOX7_5 ' on both progenitor genomes and the tetraploid. Amplicons cover partial E2, partial E4, entire E3, I2, and I3. b . Primers 1991/1992 amplified AhPLD2 in tetraploid and diploid progenitor species; primers 2120/2121 amplified AhPLD1 in both sub-genomes; primers 2187/2188 amplified AhLOX7_3 ' in both sub-genomes, amplicons contain partial E7, E9, entire E8, I7, and I8; primers 2186/2188 amplified AhLOX7_3 ' in both sub-genomes at a slightly different amplification start position resulted in similar amplification. DNA size standard: All-purpose Hi-Lo DNA marker, Bionexus, catalogue no. BN2050. TR = A. hypogaea cv. Tifrunner, Ad- A. duranensis (A genome), Ai = A. ipaensis (B genome).
    Figure Legend Snippet: PCR amplicons of AhLOX7 _5', AhLOX7_3 ', AhPLD1 , and AhPLD2. PCR amplification of AhLOX7_5 ', AhLOX7 _3', AhPLD1 , and AhPLD2 . a . Primers 2199/2184 amplify AhLOX7_5 ' on both progenitor genomes and the tetraploid. Amplicons cover partial E2, partial E4, entire E3, I2, and I3. b . Primers 1991/1992 amplified AhPLD2 in tetraploid and diploid progenitor species; primers 2120/2121 amplified AhPLD1 in both sub-genomes; primers 2187/2188 amplified AhLOX7_3 ' in both sub-genomes, amplicons contain partial E7, E9, entire E8, I7, and I8; primers 2186/2188 amplified AhLOX7_3 ' in both sub-genomes at a slightly different amplification start position resulted in similar amplification. DNA size standard: All-purpose Hi-Lo DNA marker, Bionexus, catalogue no. BN2050. TR = A. hypogaea cv. Tifrunner, Ad- A. duranensis (A genome), Ai = A. ipaensis (B genome).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    7) Product Images from "TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)"

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1348-0

    PCR amplicons of AhLOX7 _5', AhLOX7_3 ', AhPLD1 , and AhPLD2. PCR amplification of AhLOX7_5 ', AhLOX7 _3', AhPLD1 , and AhPLD2 . a . Primers 2199/2184 amplify AhLOX7_5 ' on both progenitor genomes and the tetraploid. Amplicons cover partial E2, partial E4, entire E3, I2, and I3. b . Primers 1991/1992 amplified AhPLD2 in tetraploid and diploid progenitor species; primers 2120/2121 amplified AhPLD1 in both sub-genomes; primers 2187/2188 amplified AhLOX7_3 ' in both sub-genomes, amplicons contain partial E7, E9, entire E8, I7, and I8; primers 2186/2188 amplified AhLOX7_3 ' in both sub-genomes at a slightly different amplification start position resulted in similar amplification. DNA size standard: All-purpose Hi-Lo DNA marker, Bionexus, catalogue no. BN2050. TR = A. hypogaea cv. Tifrunner, Ad- A. duranensis (A genome), Ai = A. ipaensis (B genome).
    Figure Legend Snippet: PCR amplicons of AhLOX7 _5', AhLOX7_3 ', AhPLD1 , and AhPLD2. PCR amplification of AhLOX7_5 ', AhLOX7 _3', AhPLD1 , and AhPLD2 . a . Primers 2199/2184 amplify AhLOX7_5 ' on both progenitor genomes and the tetraploid. Amplicons cover partial E2, partial E4, entire E3, I2, and I3. b . Primers 1991/1992 amplified AhPLD2 in tetraploid and diploid progenitor species; primers 2120/2121 amplified AhPLD1 in both sub-genomes; primers 2187/2188 amplified AhLOX7_3 ' in both sub-genomes, amplicons contain partial E7, E9, entire E8, I7, and I8; primers 2186/2188 amplified AhLOX7_3 ' in both sub-genomes at a slightly different amplification start position resulted in similar amplification. DNA size standard: All-purpose Hi-Lo DNA marker, Bionexus, catalogue no. BN2050. TR = A. hypogaea cv. Tifrunner, Ad- A. duranensis (A genome), Ai = A. ipaensis (B genome).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    8) Product Images from "Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as Vectors of Leishmania infantum in Tbilisi, Georgia"

    Article Title: Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as Vectors of Leishmania infantum in Tbilisi, Georgia

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001609

    Analysis of blood meals from wild-caught sand flies in the Tbilisi VL focus. Cyanine-stained agarose gel showing (A) dog- or human-specific and (B) universal cytochrome b amplicons from polymerase chain reactions containing DNA extracted from blood-fed phlebotomine sand flies. Control products are shown in lane 1 ( Phlebotomus perniciousus fed on a dog) and 2 ( Phlebotomus dubosqi fed on a human). Products of representative samples of field-collected blood-fed Phlebotomus kandelakii are shown in lanes 3–6. Lane 7, negative control. The outside lanes are 100 basepair DNA ladders. bp = basepairs.
    Figure Legend Snippet: Analysis of blood meals from wild-caught sand flies in the Tbilisi VL focus. Cyanine-stained agarose gel showing (A) dog- or human-specific and (B) universal cytochrome b amplicons from polymerase chain reactions containing DNA extracted from blood-fed phlebotomine sand flies. Control products are shown in lane 1 ( Phlebotomus perniciousus fed on a dog) and 2 ( Phlebotomus dubosqi fed on a human). Products of representative samples of field-collected blood-fed Phlebotomus kandelakii are shown in lanes 3–6. Lane 7, negative control. The outside lanes are 100 basepair DNA ladders. bp = basepairs.

    Techniques Used: Staining, Agarose Gel Electrophoresis, Negative Control

    9) Product Images from "A Feeding Induced Switch from a Variable to a Homogenous State of the Earthworm Gut Microbiota within a Host Population"

    Article Title: A Feeding Induced Switch from a Variable to a Homogenous State of the Earthworm Gut Microbiota within a Host Population

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007528

    Total bacterial diversity associated with earthworms determined by 16S rRNA gene clone library analyses. PCA plot (A) and dendrogram (B) representation of all the bacterial 16S rRNA gene sequences (n = 1000) determined in this work. In the PCA plot, the major bacterial groups identified are marked with solid lines, while the selectivity of the amplicons used ( Table S1 ) are marked with color-code stippled lines. Corresponding color-coding is given for the dendrogram. The PCA color-coding represents the respective libraries; red – whole earthworm, brown – gut-dissected earthworm, and blue –bedding/feed. The PCA axis numbering represents the respective AIBIMM coordinates.
    Figure Legend Snippet: Total bacterial diversity associated with earthworms determined by 16S rRNA gene clone library analyses. PCA plot (A) and dendrogram (B) representation of all the bacterial 16S rRNA gene sequences (n = 1000) determined in this work. In the PCA plot, the major bacterial groups identified are marked with solid lines, while the selectivity of the amplicons used ( Table S1 ) are marked with color-code stippled lines. Corresponding color-coding is given for the dendrogram. The PCA color-coding represents the respective libraries; red – whole earthworm, brown – gut-dissected earthworm, and blue –bedding/feed. The PCA axis numbering represents the respective AIBIMM coordinates.

    Techniques Used:

    10) Product Images from "Capturing in situ Virus-Host Range and Interaction Dynamics through Gene Fusion with epicPCR"

    Article Title: Capturing in situ Virus-Host Range and Interaction Dynamics through Gene Fusion with epicPCR

    Journal: bioRxiv

    doi: 10.1101/2020.08.14.250803

    EpicPCR identifies phage-host interactions in the environment without cultivation. A) Overview of the experimental design for epicPCR, identifying phage-host interactions through single-cell isolation in emulsion droplets and virus and host marker gene fusion. Left: individual cells are isolated within emulsion droplets and the genome of the host (blue circle) and virus (red curved lines) serve as the template for the gene fusion reaction. Middle: fusion PCR joins and amplifies viral and host marker genes from actively infected cells within emulsion droplets. RNR (ribonucleotide reductase; red) and 16S (16S rRNA gene; blue) are joined through an overlapping primer sequence (light blue). Right: fused amplicons are sequenced and analyzed to identify the network of viral-host interactions in the environment. Overall, 95 interactions (red cells) between 40 unique phage RNR sequences and 27 unique host 16S rRNA sequences were identified. B-C) Specificity of the method was tested by spiking Rhode River water samples with a mock community or single uninfected host ( E. coli) prior to emulsification. B) Proportion of fusion sequences belonging to uninfected mock (red) and Rhode River (blue) community members as a function of their community abundance. Uninfected mock community sequences were not found to be associated within any fusion products. C) Proportion of fusion products containing E. coli when spiked into Rhode River samples at 0.1% (n = 17), 1% (n = 17), and 5% (n = 7) of the community. Post-filtered sequences are those interactions observed in a minimum of three libraries.
    Figure Legend Snippet: EpicPCR identifies phage-host interactions in the environment without cultivation. A) Overview of the experimental design for epicPCR, identifying phage-host interactions through single-cell isolation in emulsion droplets and virus and host marker gene fusion. Left: individual cells are isolated within emulsion droplets and the genome of the host (blue circle) and virus (red curved lines) serve as the template for the gene fusion reaction. Middle: fusion PCR joins and amplifies viral and host marker genes from actively infected cells within emulsion droplets. RNR (ribonucleotide reductase; red) and 16S (16S rRNA gene; blue) are joined through an overlapping primer sequence (light blue). Right: fused amplicons are sequenced and analyzed to identify the network of viral-host interactions in the environment. Overall, 95 interactions (red cells) between 40 unique phage RNR sequences and 27 unique host 16S rRNA sequences were identified. B-C) Specificity of the method was tested by spiking Rhode River water samples with a mock community or single uninfected host ( E. coli) prior to emulsification. B) Proportion of fusion sequences belonging to uninfected mock (red) and Rhode River (blue) community members as a function of their community abundance. Uninfected mock community sequences were not found to be associated within any fusion products. C) Proportion of fusion products containing E. coli when spiked into Rhode River samples at 0.1% (n = 17), 1% (n = 17), and 5% (n = 7) of the community. Post-filtered sequences are those interactions observed in a minimum of three libraries.

    Techniques Used: Single-cell Isolation, Marker, Isolation, Polymerase Chain Reaction, Infection, Sequencing

    11) Product Images from "Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1"

    Article Title: Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

    Journal: Journal of Insect Science

    doi: 10.1093/jisesa/iev137

    Derivative dissociation curves and difference plots produced by the control and species-specific amplicons produced using optimized primer combination and squish buffer lysates from legs of H. armigera , H. zea , and 1:24 ratio of H. armigera : H. zea legs. KAPA YBR Fast Master Mix was used for DNA amplification. (A) Dissociation curves of H. armigera (red) and H. zea (blue) have the peaks from control amplicon and the species-specific amplicon. Mixed DNA (green) has all three peaks. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.
    Figure Legend Snippet: Derivative dissociation curves and difference plots produced by the control and species-specific amplicons produced using optimized primer combination and squish buffer lysates from legs of H. armigera , H. zea , and 1:24 ratio of H. armigera : H. zea legs. KAPA YBR Fast Master Mix was used for DNA amplification. (A) Dissociation curves of H. armigera (red) and H. zea (blue) have the peaks from control amplicon and the species-specific amplicon. Mixed DNA (green) has all three peaks. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.

    Techniques Used: Produced, Amplification, Generated, Software

    Derivative dissociation curves and difference plots produced by the control and species-specific amplicons of H. armigera , H. zea , Helio. subflexa, and Helio. virescens . (A) Derivative dissociation curves of H. armigera (red) and H. zea (blue), Helio. subflexa (green), and Helio. virescens (purple). The peaks produced by 18S rRNA subunit amplicon (∼78°C) is present in all species. Dissociation curves of H. armigera and H. zea also contains peaks specific to each species. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.
    Figure Legend Snippet: Derivative dissociation curves and difference plots produced by the control and species-specific amplicons of H. armigera , H. zea , Helio. subflexa, and Helio. virescens . (A) Derivative dissociation curves of H. armigera (red) and H. zea (blue), Helio. subflexa (green), and Helio. virescens (purple). The peaks produced by 18S rRNA subunit amplicon (∼78°C) is present in all species. Dissociation curves of H. armigera and H. zea also contains peaks specific to each species. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.

    Techniques Used: Produced, Amplification, Generated, Software

    Gel images of ITS1 amplicons from a representative set of DNA amplified using the oligonucleotide primer mix developed for species detection. (A) Amplicons produced with common (3373) primer, and primers specific to H. armigera (3374) and H. zea (3377) and 0.4 μM final concentration. Lanes 1–3, 5, 8, and 9: 147 bp amplicon of H. armigera ; Lanes 10–14: 314 bp amplicon of H. zea ; Lane 4: F1 male hybrid of H. zea and H. armigera ; Lane 6: a mixture of H. zea and H. armigera DNA. (B) Amplicons produced with optimal concentrations of primers for species-specific amplicons and the 83 bp 18S rRNA subunit control amplicon. Lanes 2 and 3: H. assulta ; Lanes 4 and 5: Helio. subflexa ; Lanes 6 and 7: Helio. virescens ; Lanes 8 and 9: H. armigera ; Lanes 10 and 11: H. zea ; Lanes 12 and 13: mixed H. armigera and H. zea DNA. M: 2-log DNA ladder (New England Biolabs, Ipswich, MA) with major DNA band sizes shown in base pairs.
    Figure Legend Snippet: Gel images of ITS1 amplicons from a representative set of DNA amplified using the oligonucleotide primer mix developed for species detection. (A) Amplicons produced with common (3373) primer, and primers specific to H. armigera (3374) and H. zea (3377) and 0.4 μM final concentration. Lanes 1–3, 5, 8, and 9: 147 bp amplicon of H. armigera ; Lanes 10–14: 314 bp amplicon of H. zea ; Lane 4: F1 male hybrid of H. zea and H. armigera ; Lane 6: a mixture of H. zea and H. armigera DNA. (B) Amplicons produced with optimal concentrations of primers for species-specific amplicons and the 83 bp 18S rRNA subunit control amplicon. Lanes 2 and 3: H. assulta ; Lanes 4 and 5: Helio. subflexa ; Lanes 6 and 7: Helio. virescens ; Lanes 8 and 9: H. armigera ; Lanes 10 and 11: H. zea ; Lanes 12 and 13: mixed H. armigera and H. zea DNA. M: 2-log DNA ladder (New England Biolabs, Ipswich, MA) with major DNA band sizes shown in base pairs.

    Techniques Used: Amplification, Produced, Concentration Assay

    Derivative dissociation plots produced by different reagents using one microliter of lysate produced by homogenizing one H. armigera leg with 24 H. zea legs in 625 μl of modified squish buffer. (A) Amplification reactions made with KAPA SYBR Fast Master Mix (purple) and NEB LongAmp Taq polymerase and buffer with 2.5 mM MgCl2 (Red). Peaks representing 18S rRNA control amplicon and species-specific amplicons of H. armigera and H. zea are present in all reactions, but melting temperatures were different in two reagents. (B) An enlarged view of the derivative dissociation plot produced by amplification reactions with NEB LongAmp Taq polymerase and buffer. Although variations in dissociation plots between samples were observed, the 3-peak dissociation plot was clearly identifiable.
    Figure Legend Snippet: Derivative dissociation plots produced by different reagents using one microliter of lysate produced by homogenizing one H. armigera leg with 24 H. zea legs in 625 μl of modified squish buffer. (A) Amplification reactions made with KAPA SYBR Fast Master Mix (purple) and NEB LongAmp Taq polymerase and buffer with 2.5 mM MgCl2 (Red). Peaks representing 18S rRNA control amplicon and species-specific amplicons of H. armigera and H. zea are present in all reactions, but melting temperatures were different in two reagents. (B) An enlarged view of the derivative dissociation plot produced by amplification reactions with NEB LongAmp Taq polymerase and buffer. Although variations in dissociation plots between samples were observed, the 3-peak dissociation plot was clearly identifiable.

    Techniques Used: Produced, Modification, Amplification

    12) Product Images from "Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958–1976"

    Article Title: Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958–1976

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2016.00091

    Results of RT-PCR and sequencing of astrovirus RNA extracted from case 3466 . A scheme of the BoAstV CH13 genome is presented. The viral genome is organized in three open-reading frames (ORF). The red bar indicates the target sequence for RT-PCR using primers MA2/MA4 within ORF 1b, which encodes for the RNA-dependent RNA polymerase. The blue and black bars show target sequences of in situ hybridization probes A and B, respectively, within ORF2 that encodes for structural capsid proteins. The nested RT-PCR protocol with primers bAV3/bAV4 (first round) and bAV1/bAV2 (second round) was designed to yield amplicons in the target region of ISH probe B (black bar). Sequence comparisons of the MA2/MA4 and of the bAV1/bAV2 amplicons of case 3466 with the BoAstV CH13 reference sequence (GenBank accession number: NC_024498) are shown. Alignments were generated with the Geneious R9 software, version 9.0.4 (Biomatters).
    Figure Legend Snippet: Results of RT-PCR and sequencing of astrovirus RNA extracted from case 3466 . A scheme of the BoAstV CH13 genome is presented. The viral genome is organized in three open-reading frames (ORF). The red bar indicates the target sequence for RT-PCR using primers MA2/MA4 within ORF 1b, which encodes for the RNA-dependent RNA polymerase. The blue and black bars show target sequences of in situ hybridization probes A and B, respectively, within ORF2 that encodes for structural capsid proteins. The nested RT-PCR protocol with primers bAV3/bAV4 (first round) and bAV1/bAV2 (second round) was designed to yield amplicons in the target region of ISH probe B (black bar). Sequence comparisons of the MA2/MA4 and of the bAV1/bAV2 amplicons of case 3466 with the BoAstV CH13 reference sequence (GenBank accession number: NC_024498) are shown. Alignments were generated with the Geneious R9 software, version 9.0.4 (Biomatters).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, In Situ Hybridization, Generated, Software

    13) Product Images from "Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta"

    Article Title: Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta

    Journal: eLife

    doi: 10.7554/eLife.56193

    Engineered cycloheximide resistance establishes genome editing conditions. ( A ) The design of a cycloheximide resistant allele, rpl36a P56Q , in S. rosetta . The protospacer adjacent motif (PAM, orange) next to the 56 th codon of rpl36a (Target, cyan), which is located on the second exon (thick black line labeled 2), provides a suitable site to design a gRNA that targets Sp Cas9 cleavage (sequence is shown underneath the locus schematic, and carets indicate the target cleavage site). A repair oligonucleotide (black line with knob) introduces a cycloheximide resistant allele, rpl36a P56Q (Mutation, purple), flanked by 100 bases of homologous sequence. The sequence of the edited allele is shown below. ( B–D ) A comparison of genotypes from populations of unedited cells ( B ), edited cells ( C ), and a strain established from a clonal isolate of edited cells ( D ) shows that cycloheximide selection enriches for rpl36a P56Q . The genotype for each population was determined by amplifying the locus with primers surrounding the editing site (black arrows in panel A) that did not overlap in sequence with the repair oligonucleotide. One of the primers had a T3 primer binding site for Sanger sequencing of amplicons (black arrow with flap). Remarkably, after selection, the wild-type allele was not detected ( B ). ( E ) S. rosetta uses repair oligonucleotides with > 20 nt homology arms for genome editing. Truncations of repair oligonucleotides encoding the rpl36a P56Q allele were designed in the same orientation as gRNAs (sense, black dots and lines) or the opposite orientation (antisense, gray dots and lines). 24 hr after S. rosetta recovered from transfections with repair templates and Sp Cas9 RNPs, cycloheximide was added to grow cells in selective media for five days, at which time the cells were harvested for counting cell density and for genotyping. Closed circles indicate that the consensus genotype of the cell population had the rpl36a P56Q allele in Sanger sequencing; whereas, open circles indicate that the cell population had the wild-type allele. E’ and E’’ show two independent trials. Notably, we observed a slight bias for repair oligonucleotides in the sense direction, particularly with shorter homology arms of 20-30 bases. Because repair templates in the sense orientation with 40–80 bases of homologous sequence resulted in the best editing, we performed subsequent optimization with a sense repair oligonucleotide that 50-base homology arms on each side of the double-stranded break. ( F ) Small quantities of Sp Cas9 RNPs are sufficient to initiate genome editing. Decreasing concentrations of Sp Cas9 RNP ( Sp Cas9 was the limiting factor) and a constant amount of repair template were transfected into S. rosetta . After characterizing genome editing outcomes by counting cell density and sequencing the consensus genotype (described in panel E), we found that low concentrations of Sp Cas9 (20 pmol) were sufficient to introduce the rpl36a P56Q allele. F’ and F’’ show two independent trials. ( G ) High concentrations of repair oligonucleotides increase genome editing efficiency. A serial dilution of a repair template was delivered into S. rosetta . The cell density and consensus genotypes from these experiments show that all concentrations of repair template can introduce the rpl36a P56Q allele, but the higher cell densities recovered after transfection with increasing concentrations of repair templates indicate more efficient editing. G’ and G’’ show two independent trials. ( H ) The addition of gRNAs stimulates genome editing. Genome editing was performed by delivering a repair oligonucleotide with Sp Cas9 without the addition of any gRNA or with a gRNA that was prepared from in vitro transcriptions (noted as gRNA in figure) or with a synthetic crRNA that was annealed to a synthetic tracrRNA (noted as crRNA). The consensus genotype and cell densities from these experiments show that gRNAs are necessary for editing and that gRNAs from either source were sufficient for editing. The dots show two independent experiments and lines show their average result.
    Figure Legend Snippet: Engineered cycloheximide resistance establishes genome editing conditions. ( A ) The design of a cycloheximide resistant allele, rpl36a P56Q , in S. rosetta . The protospacer adjacent motif (PAM, orange) next to the 56 th codon of rpl36a (Target, cyan), which is located on the second exon (thick black line labeled 2), provides a suitable site to design a gRNA that targets Sp Cas9 cleavage (sequence is shown underneath the locus schematic, and carets indicate the target cleavage site). A repair oligonucleotide (black line with knob) introduces a cycloheximide resistant allele, rpl36a P56Q (Mutation, purple), flanked by 100 bases of homologous sequence. The sequence of the edited allele is shown below. ( B–D ) A comparison of genotypes from populations of unedited cells ( B ), edited cells ( C ), and a strain established from a clonal isolate of edited cells ( D ) shows that cycloheximide selection enriches for rpl36a P56Q . The genotype for each population was determined by amplifying the locus with primers surrounding the editing site (black arrows in panel A) that did not overlap in sequence with the repair oligonucleotide. One of the primers had a T3 primer binding site for Sanger sequencing of amplicons (black arrow with flap). Remarkably, after selection, the wild-type allele was not detected ( B ). ( E ) S. rosetta uses repair oligonucleotides with > 20 nt homology arms for genome editing. Truncations of repair oligonucleotides encoding the rpl36a P56Q allele were designed in the same orientation as gRNAs (sense, black dots and lines) or the opposite orientation (antisense, gray dots and lines). 24 hr after S. rosetta recovered from transfections with repair templates and Sp Cas9 RNPs, cycloheximide was added to grow cells in selective media for five days, at which time the cells were harvested for counting cell density and for genotyping. Closed circles indicate that the consensus genotype of the cell population had the rpl36a P56Q allele in Sanger sequencing; whereas, open circles indicate that the cell population had the wild-type allele. E’ and E’’ show two independent trials. Notably, we observed a slight bias for repair oligonucleotides in the sense direction, particularly with shorter homology arms of 20-30 bases. Because repair templates in the sense orientation with 40–80 bases of homologous sequence resulted in the best editing, we performed subsequent optimization with a sense repair oligonucleotide that 50-base homology arms on each side of the double-stranded break. ( F ) Small quantities of Sp Cas9 RNPs are sufficient to initiate genome editing. Decreasing concentrations of Sp Cas9 RNP ( Sp Cas9 was the limiting factor) and a constant amount of repair template were transfected into S. rosetta . After characterizing genome editing outcomes by counting cell density and sequencing the consensus genotype (described in panel E), we found that low concentrations of Sp Cas9 (20 pmol) were sufficient to introduce the rpl36a P56Q allele. F’ and F’’ show two independent trials. ( G ) High concentrations of repair oligonucleotides increase genome editing efficiency. A serial dilution of a repair template was delivered into S. rosetta . The cell density and consensus genotypes from these experiments show that all concentrations of repair template can introduce the rpl36a P56Q allele, but the higher cell densities recovered after transfection with increasing concentrations of repair templates indicate more efficient editing. G’ and G’’ show two independent trials. ( H ) The addition of gRNAs stimulates genome editing. Genome editing was performed by delivering a repair oligonucleotide with Sp Cas9 without the addition of any gRNA or with a gRNA that was prepared from in vitro transcriptions (noted as gRNA in figure) or with a synthetic crRNA that was annealed to a synthetic tracrRNA (noted as crRNA). The consensus genotype and cell densities from these experiments show that gRNAs are necessary for editing and that gRNAs from either source were sufficient for editing. The dots show two independent experiments and lines show their average result.

    Techniques Used: Labeling, Sequencing, Mutagenesis, Selection, Binding Assay, Transfection, Introduce, Serial Dilution, In Vitro

    14) Product Images from "A Pilot Comparative Study Between Serological and Genetic Investigations in Relationship to Clinical Outcomes on Patients with Cystic Echinococcosis"

    Article Title: A Pilot Comparative Study Between Serological and Genetic Investigations in Relationship to Clinical Outcomes on Patients with Cystic Echinococcosis

    Journal: Helminthologia

    doi: 10.2478/helm-2020-0012

    CO1, NAD1 and ITS-1 Gene Amplicons. A. CO1 gene amplicons (446 bp). M:DNA Marker, 1:Positive Control, 2-3:Human Isolates, 4:Negative Control (Distilled water) B. NAD1 gene amplicons (378 bp). M:DNA Marker, 1:Positive Control, 2:Negative Control (Distilled water), 3-4:Human Isolates. C. ITS-1 gene amplicons. M:DNA Marker, 1:Positive Control, 2:Negative Control (Distilled water), 3:Human.
    Figure Legend Snippet: CO1, NAD1 and ITS-1 Gene Amplicons. A. CO1 gene amplicons (446 bp). M:DNA Marker, 1:Positive Control, 2-3:Human Isolates, 4:Negative Control (Distilled water) B. NAD1 gene amplicons (378 bp). M:DNA Marker, 1:Positive Control, 2:Negative Control (Distilled water), 3-4:Human Isolates. C. ITS-1 gene amplicons. M:DNA Marker, 1:Positive Control, 2:Negative Control (Distilled water), 3:Human.

    Techniques Used: Marker, Positive Control, Negative Control

    15) Product Images from "Time-Resolved Tracking of Mutations Reveals Diverse Allele Dynamics during Escherichia coli Antimicrobial Adaptive Evolution to Single Drugs and Drug Pairs"

    Article Title: Time-Resolved Tracking of Mutations Reveals Diverse Allele Dynamics during Escherichia coli Antimicrobial Adaptive Evolution to Single Drugs and Drug Pairs

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00893

    Experimental overview of the investigation from sample collection description, adaptation laboratory evolution (ALE) and amplicon frequency workflow. (A) Background for our frozen sample conditions showing five drug conditions (AMK, CHL, CIP, AMK–CHL, and CHL–CIP) each with three lineages (Lineages A, B, C) covering six time points (days 2, 4, 6, 8, 10, and 12) for the laboratory adaptation experiments. (B) The workflow used to assess different loci from different drug conditions, lineages, and time points in which (1) the DNA was extracted from the different samples, (2) each sample has amplicons for each locus generated by primers that had a DNA amplifying region and an Illumina Nextera XT compatible overhang, (3) these were then barcoded using indices from the Illumina Nextera XT library preparation kit, (4) all of the generated amplicons were pooled together based on fragment size and concentration, (5) the pooled amplicon library was sequenced on the MiSeq Illumina sequencing platform, (6) each sample was sorted by barcode, and each was used to generate a file to map to the reference genome and call variants in the bacterial population and to calculate their frequencies.
    Figure Legend Snippet: Experimental overview of the investigation from sample collection description, adaptation laboratory evolution (ALE) and amplicon frequency workflow. (A) Background for our frozen sample conditions showing five drug conditions (AMK, CHL, CIP, AMK–CHL, and CHL–CIP) each with three lineages (Lineages A, B, C) covering six time points (days 2, 4, 6, 8, 10, and 12) for the laboratory adaptation experiments. (B) The workflow used to assess different loci from different drug conditions, lineages, and time points in which (1) the DNA was extracted from the different samples, (2) each sample has amplicons for each locus generated by primers that had a DNA amplifying region and an Illumina Nextera XT compatible overhang, (3) these were then barcoded using indices from the Illumina Nextera XT library preparation kit, (4) all of the generated amplicons were pooled together based on fragment size and concentration, (5) the pooled amplicon library was sequenced on the MiSeq Illumina sequencing platform, (6) each sample was sorted by barcode, and each was used to generate a file to map to the reference genome and call variants in the bacterial population and to calculate their frequencies.

    Techniques Used: Amplification, Generated, Concentration Assay, Sequencing

    16) Product Images from "A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function"

    Article Title: A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24036-4

    duoxa germline mutants develop goitrous thyroid phenotypes. ( A ) Zebrafish duoxa genomic locus on chromosome 25 with sequences for the wild-type (WT) allele and a mutant allele ( duoxa Δ11) containing a 11 bp deletion in exon 2. The sgRNA target site is underlined in the WT sequence. ( B ) PCR analysis of genomic DNA allows for sensitive detection of WT and duoxa Δ11 mutant alleles in individual fish (F3 generation). Polyacrylamide gel electrophoresis of PCR amplicons of WT, heterozygous and homozygous duoxa Δ11 carriers. Full-length gel is shown in Supplementary Fig. 7 from which lanes 1, 2, 3, 4 and 6 are shown in the cropped gel image. ( C ) Thyroid phenotyping of duoxa Δ11 mutant fish maintained on a Tg ( tg:nlsEGFP ) background. Immunofluorescence staining (GFP and T4) of 6 dpf larvae (ventral view, anterior to the top, scale bars: 20 µm) showed goitrous thyroid enlargement and absence of detectable T4 staining in all homozygous duoxa Δ11 fish ( N = 30). Larvae with a normal-looking thyroid morphology ( N = 30) were genotyped as either WT or heterozygous carriers of the duoxa Δ11 allele. For each larvae shown, 3.5-fold magnified views of the thyroid region are displayed (merge of GFP/T4 and T4 only). ( D ) Proportion of larvae with goitrous thyroid phenotype as detected in the progeny of three independent inbreeding experiments with heterozygous duoxa Δ11 fish.
    Figure Legend Snippet: duoxa germline mutants develop goitrous thyroid phenotypes. ( A ) Zebrafish duoxa genomic locus on chromosome 25 with sequences for the wild-type (WT) allele and a mutant allele ( duoxa Δ11) containing a 11 bp deletion in exon 2. The sgRNA target site is underlined in the WT sequence. ( B ) PCR analysis of genomic DNA allows for sensitive detection of WT and duoxa Δ11 mutant alleles in individual fish (F3 generation). Polyacrylamide gel electrophoresis of PCR amplicons of WT, heterozygous and homozygous duoxa Δ11 carriers. Full-length gel is shown in Supplementary Fig. 7 from which lanes 1, 2, 3, 4 and 6 are shown in the cropped gel image. ( C ) Thyroid phenotyping of duoxa Δ11 mutant fish maintained on a Tg ( tg:nlsEGFP ) background. Immunofluorescence staining (GFP and T4) of 6 dpf larvae (ventral view, anterior to the top, scale bars: 20 µm) showed goitrous thyroid enlargement and absence of detectable T4 staining in all homozygous duoxa Δ11 fish ( N = 30). Larvae with a normal-looking thyroid morphology ( N = 30) were genotyped as either WT or heterozygous carriers of the duoxa Δ11 allele. For each larvae shown, 3.5-fold magnified views of the thyroid region are displayed (merge of GFP/T4 and T4 only). ( D ) Proportion of larvae with goitrous thyroid phenotype as detected in the progeny of three independent inbreeding experiments with heterozygous duoxa Δ11 fish.

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Polyacrylamide Gel Electrophoresis, Immunofluorescence, Staining

    17) Product Images from "Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola ▿"

    Article Title: Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola ▿

    Journal:

    doi: 10.1128/JB.00046-08

    Conservation of AtcS and AtcR among T. denticola strains. PCR amplicons obtained with atcS and atcR gene-specific primer sets from seven different strains are shown in panel A. Lane NT shows PCRs with no template added. Western blotting analysis of AtcS
    Figure Legend Snippet: Conservation of AtcS and AtcR among T. denticola strains. PCR amplicons obtained with atcS and atcR gene-specific primer sets from seven different strains are shown in panel A. Lane NT shows PCRs with no template added. Western blotting analysis of AtcS

    Techniques Used: Polymerase Chain Reaction, Western Blot

    RT-PCR and Northern blot analysis of the atcR/atcS locus. Panel A shows a schematic of atcR-atcS and their up- and downstream genes (TIGR annotation). The lengths of the intergenic regions are shown in parentheses. Primer binding sites and the amplicons
    Figure Legend Snippet: RT-PCR and Northern blot analysis of the atcR/atcS locus. Panel A shows a schematic of atcR-atcS and their up- and downstream genes (TIGR annotation). The lengths of the intergenic regions are shown in parentheses. Primer binding sites and the amplicons

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Binding Assay

    18) Product Images from "Analysis of the Type IV Fimbrial-Subunit Gene fimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths"

    Article Title: Analysis of the Type IV Fimbrial-Subunit Gene fimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.67.2.598-607.2001

    Specificity of the PCR with nested primers developed by using the internal sequence of fimA from X. campestris pv. vesicatoria NCPPB3240: analysis of isolates of X. vesicatoria and X. axonopodis pv. vesicatoria . Lane O, negative control; lane 1, X. vesicatoria LMG911; lane 2, X. vesicatoria LMG917; lane 3, X. axonopodis pv. vesicatoria LMG668; lane 4, X. axonopodis pv. vesicatoria LMG905; lane 5, X. vesicatoria LMG920; lane 6, X. vesicatoria LMG925; lane 7, X. vesicatoria NCCPB3240; lane 8, X. axonopodis pv. vesicatoria LMG910; lane 9, X. axonopodis pv. vesicatoria LMG913; lane 10, X. axonopodis pv. vesicatoria LMG922; lane 11, X. axonopodis pv. vesicatoria LMG929; lane 12, X. vesicatoria ATCC 35937; lane 13, X. vesicatoria ATCC 11551; lanes M, 100-bp ladder (Promega). The arrow indicates 202-bp X. vesicatoria -specific amplicons.
    Figure Legend Snippet: Specificity of the PCR with nested primers developed by using the internal sequence of fimA from X. campestris pv. vesicatoria NCPPB3240: analysis of isolates of X. vesicatoria and X. axonopodis pv. vesicatoria . Lane O, negative control; lane 1, X. vesicatoria LMG911; lane 2, X. vesicatoria LMG917; lane 3, X. axonopodis pv. vesicatoria LMG668; lane 4, X. axonopodis pv. vesicatoria LMG905; lane 5, X. vesicatoria LMG920; lane 6, X. vesicatoria LMG925; lane 7, X. vesicatoria NCCPB3240; lane 8, X. axonopodis pv. vesicatoria LMG910; lane 9, X. axonopodis pv. vesicatoria LMG913; lane 10, X. axonopodis pv. vesicatoria LMG922; lane 11, X. axonopodis pv. vesicatoria LMG929; lane 12, X. vesicatoria ATCC 35937; lane 13, X. vesicatoria ATCC 11551; lanes M, 100-bp ladder (Promega). The arrow indicates 202-bp X. vesicatoria -specific amplicons.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control

    19) Product Images from "Anatoxin-a Synthetase Gene Cluster of the Cyanobacterium Anabaena sp. Strain 37 and Molecular Methods To Detect Potential Producers ▿ sp. Strain 37 and Molecular Methods To Detect Potential Producers ▿ †"

    Article Title: Anatoxin-a Synthetase Gene Cluster of the Cyanobacterium Anabaena sp. Strain 37 and Molecular Methods To Detect Potential Producers ▿ sp. Strain 37 and Molecular Methods To Detect Potential Producers ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.06022-11

    RFLP analysis of anaC amplicons (366 bp) of cyanobacterial strains and environmental samples with the HhaI and HinfI restriction enzymes. The amplicons are indicated above the lanes according to the strain/sample used for amplification: Anabaena sp. 37
    Figure Legend Snippet: RFLP analysis of anaC amplicons (366 bp) of cyanobacterial strains and environmental samples with the HhaI and HinfI restriction enzymes. The amplicons are indicated above the lanes according to the strain/sample used for amplification: Anabaena sp. 37

    Techniques Used: Environmental Sampling, Amplification

    20) Product Images from "Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo"

    Article Title: Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo

    Journal: Journal of Virology

    doi: 10.1128/JVI.02420-15

    Alteration of Cx43 in mouse brain due to MHV-A59 infection. Mice were infected intracranially with MHV-A59 at 50% of the LD 50 or with PBS-BSA for mock-infected mice. Mice were sacrificed at day 5 p.i., after which their liver and brain tissues were processed for RNA extraction. cDNA was synthesized from RNA of the brains and livers. To confirm infection, cDNAs from liver were amplified for virus-specific antinucleocapsid primers (IZJ5 and IZJ6). (A) Intracranial injection of mice with the virus showed the presence of nucleocapsid-specific amplicons (Infected lanes 1, 2, and 3) in liver. As expected, no such amplification was noted from mock-inoculated mice (Mock lanes 1 and 2). (B) Real-time qPCR analysis of the RNA samples from brain showed a significant 3.13- ± 0.06-fold reduction in relative abundance of Cx43 mRNA after MHV-A59 infection. The mean ± SEM incidences from three different mice are shown. (****, P
    Figure Legend Snippet: Alteration of Cx43 in mouse brain due to MHV-A59 infection. Mice were infected intracranially with MHV-A59 at 50% of the LD 50 or with PBS-BSA for mock-infected mice. Mice were sacrificed at day 5 p.i., after which their liver and brain tissues were processed for RNA extraction. cDNA was synthesized from RNA of the brains and livers. To confirm infection, cDNAs from liver were amplified for virus-specific antinucleocapsid primers (IZJ5 and IZJ6). (A) Intracranial injection of mice with the virus showed the presence of nucleocapsid-specific amplicons (Infected lanes 1, 2, and 3) in liver. As expected, no such amplification was noted from mock-inoculated mice (Mock lanes 1 and 2). (B) Real-time qPCR analysis of the RNA samples from brain showed a significant 3.13- ± 0.06-fold reduction in relative abundance of Cx43 mRNA after MHV-A59 infection. The mean ± SEM incidences from three different mice are shown. (****, P

    Techniques Used: Infection, Mouse Assay, RNA Extraction, Synthesized, Amplification, Injection, Real-time Polymerase Chain Reaction

    21) Product Images from "Occurrence and diversity of avian haemosporidia in Afrotropical landbirds"

    Article Title: Occurrence and diversity of avian haemosporidia in Afrotropical landbirds

    Journal: International Journal for Parasitology: Parasites and Wildlife

    doi: 10.1016/j.ijppaw.2018.12.002

    Gel electrophoresis image of nested PCR of ten-fold serial dilutions of Plasmodium . relictum positive control amplicons (P5 – P01; 6.8 × 10 5 –0.68 gene copies/μl DNA). S4 – Sample from an African Pygmy Kingfisher; water - negative control.
    Figure Legend Snippet: Gel electrophoresis image of nested PCR of ten-fold serial dilutions of Plasmodium . relictum positive control amplicons (P5 – P01; 6.8 × 10 5 –0.68 gene copies/μl DNA). S4 – Sample from an African Pygmy Kingfisher; water - negative control.

    Techniques Used: Nucleic Acid Electrophoresis, Nested PCR, Positive Control, Negative Control

    22) Product Images from "Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera"

    Article Title: Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera

    Journal: Biotechnology Reports

    doi: 10.1016/j.btre.2017.05.001

    Amplification of (a) cry1 and (b) cry2 genes in representative B. thuringiensis isolates indicating amplification of 1500 bp and 700 bp amplicons, respectively. Lane M: 1 kb DNA size marker (Fermentas). 1.0% agarose/EtBr gel.
    Figure Legend Snippet: Amplification of (a) cry1 and (b) cry2 genes in representative B. thuringiensis isolates indicating amplification of 1500 bp and 700 bp amplicons, respectively. Lane M: 1 kb DNA size marker (Fermentas). 1.0% agarose/EtBr gel.

    Techniques Used: Amplification, Marker

    23) Product Images from "Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics"

    Article Title: Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006075

    Number of virus-specific amplicons detected via semiconductor sequencing in 1 million total reads with (+) or without (-) amplification with the novel panel (YFV: Yellow fever virus; RVFV: Rift Valley fever virus; CCHFV: Crimean-Congo hemorrhagic fever virus).
    Figure Legend Snippet: Number of virus-specific amplicons detected via semiconductor sequencing in 1 million total reads with (+) or without (-) amplification with the novel panel (YFV: Yellow fever virus; RVFV: Rift Valley fever virus; CCHFV: Crimean-Congo hemorrhagic fever virus).

    Techniques Used: Sequencing, Amplification

    Number of virus-specific amplicons detected via semiconductor sequencing in 1 million total reads with (+) or without (-) amplification with the novel panel (EBOV: Ebola virus; CHIKV: Chikungunya virus; JUNV: Junin virus).
    Figure Legend Snippet: Number of virus-specific amplicons detected via semiconductor sequencing in 1 million total reads with (+) or without (-) amplification with the novel panel (EBOV: Ebola virus; CHIKV: Chikungunya virus; JUNV: Junin virus).

    Techniques Used: Sequencing, Amplification

    24) Product Images from "Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo"

    Article Title: Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv034

    RLM-RACE analysis of the 5′ termini of the PSTVd (+) sgRNAs 1 to 5, using as control a PSTVd (+) ml form with 5′-hydroxyl (G2) and 2′,3′-cyclic phosphodiester (C1) termini resulting from ribozyme-mediated self-cleavage. Prior to RLM-RACE (see Supplementary Table S3 for the RNA adaptor and primers used), aliquots of each RNA were left untreated (lanes 1 and 3) or treated with PK and ATP to phosphorylate 5′-hydroxyl groups (lanes 2 and 4), with the resulting amplification products being then separated by non-denaturing PAGE and stained with ethidium bromide. Arrowheads indicate the amplicons of expected length that were eluted, cloned and sequenced. Lane M, DNA markers.
    Figure Legend Snippet: RLM-RACE analysis of the 5′ termini of the PSTVd (+) sgRNAs 1 to 5, using as control a PSTVd (+) ml form with 5′-hydroxyl (G2) and 2′,3′-cyclic phosphodiester (C1) termini resulting from ribozyme-mediated self-cleavage. Prior to RLM-RACE (see Supplementary Table S3 for the RNA adaptor and primers used), aliquots of each RNA were left untreated (lanes 1 and 3) or treated with PK and ATP to phosphorylate 5′-hydroxyl groups (lanes 2 and 4), with the resulting amplification products being then separated by non-denaturing PAGE and stained with ethidium bromide. Arrowheads indicate the amplicons of expected length that were eluted, cloned and sequenced. Lane M, DNA markers.

    Techniques Used: Amplification, Polyacrylamide Gel Electrophoresis, Staining, Clone Assay

    25) Product Images from "Isolation and characterization of bacteriocinogenic lactic bacteria from M-Tuba and Tepache, two traditional fermented beverages in México"

    Article Title: Isolation and characterization of bacteriocinogenic lactic bacteria from M-Tuba and Tepache, two traditional fermented beverages in México

    Journal: Food Science & Nutrition

    doi: 10.1002/fsn3.236

    Detection of nisin, enterocin, and pediocin genes in different bacterial strains isolated from M-Tuba and Tepache. (A) Standardization of conditions for multiplex PCR (mPCR). Mixes DNA samples from Lactococcus lactis , Enterococcus faecium , and Pediococcus acidilactici were used as positive controls to amplify nisin, enterocin, and pediocin, respectively. Lane 1, DNA Ladder 1 Kb Plus DNA (Invitrogen); lanes 2–9, amplicons obtained from positive bacteria using an alignment interval of temperature of 48.0 to 57.5°C (intervals of 57.5, 56.8, 54.5, 55.7, 53.9, 51.5, 49.9, 48.7, and 48.0 respectively). For further amplification, we select the alignment temperatures of 54.5°C, which correspond to the amplification in lane 4. (B) Multiplex PCR of isolates obtained from Tepache and M-Tuba. Lane 1, DNA Ladder 100 bp (NEB); lanes 2–7 and 10, amplicons corresponding to the enterocin gene (∼412 bp); lane 8, nisin (∼ 608 bp). (C) Identification of nisin and enterocin genes from different isolates. Lane 1, DNA Ladder 1 kb Plus DNA (Invitrogen); lane 2, amplicon of nisin from L. lactis used as positive control; lanes 3 and 4, amplicons corresponding to nisin from L. lactis TuAB1 and L. lactis TeA1; lane 5, amplicons of enterocin from E. faecium TuAB2. Similar amplicon than E. faecium TuAB2 was obtained with E. faecium TeA2 (data not shown). Confirmation that amplicons correspond to nisin and enterocin genes, was carried out by sequencing.
    Figure Legend Snippet: Detection of nisin, enterocin, and pediocin genes in different bacterial strains isolated from M-Tuba and Tepache. (A) Standardization of conditions for multiplex PCR (mPCR). Mixes DNA samples from Lactococcus lactis , Enterococcus faecium , and Pediococcus acidilactici were used as positive controls to amplify nisin, enterocin, and pediocin, respectively. Lane 1, DNA Ladder 1 Kb Plus DNA (Invitrogen); lanes 2–9, amplicons obtained from positive bacteria using an alignment interval of temperature of 48.0 to 57.5°C (intervals of 57.5, 56.8, 54.5, 55.7, 53.9, 51.5, 49.9, 48.7, and 48.0 respectively). For further amplification, we select the alignment temperatures of 54.5°C, which correspond to the amplification in lane 4. (B) Multiplex PCR of isolates obtained from Tepache and M-Tuba. Lane 1, DNA Ladder 100 bp (NEB); lanes 2–7 and 10, amplicons corresponding to the enterocin gene (∼412 bp); lane 8, nisin (∼ 608 bp). (C) Identification of nisin and enterocin genes from different isolates. Lane 1, DNA Ladder 1 kb Plus DNA (Invitrogen); lane 2, amplicon of nisin from L. lactis used as positive control; lanes 3 and 4, amplicons corresponding to nisin from L. lactis TuAB1 and L. lactis TeA1; lane 5, amplicons of enterocin from E. faecium TuAB2. Similar amplicon than E. faecium TuAB2 was obtained with E. faecium TeA2 (data not shown). Confirmation that amplicons correspond to nisin and enterocin genes, was carried out by sequencing.

    Techniques Used: Isolation, Multiplex Assay, Polymerase Chain Reaction, Amplification, Positive Control, Sequencing

    26) Product Images from "Transcriptional and phenotypical heterogeneity of Trypanosoma cruzi cell populations"

    Article Title: Transcriptional and phenotypical heterogeneity of Trypanosoma cruzi cell populations

    Journal: Open Biology

    doi: 10.1098/rsob.150190

    Analysis of MASP clonal expression by capillary electrophoresis and western blot. ( a ) In silico gel obtained by PCR fingerprinting analysing PCR products of three-step nested PCR for each isogenic line. On the top, a dendogram based on the similarity of the MASP amplicons pattern. The distances are measured at the right side of the dendogram with the percentage of similarity among the samples. ( b ) Bar graph representing the number of MASP amplicons obtained in the third step of the nested PCR for each clone and the parental strain. ( c ) Number of amplicons obtained from the second and the third steps of the nested PCR in the trypomastigotes of the parental cell line. ( d ) Frequency of bands according to their size amplified from the third nested PCR of the epimastigote stage for all the clones and from the epimastigote and trypomastigote forms of the parental cell line. ( e ) Western blot analysis of total protein extracts from the epimastigote forms of all the clones, and the epimastigote and trypomastigote stage of the parental strain using anti-MASP-Ag mouse antisera. Anti-tubulin sera were used as loading control and pre-immune sera as negative control. Below, bar graphs representing the ratio 45 kDa/tubulin and 40 kDa/tubulin. Second PCR and third PCR are the products of the second and third PCRs of the three-step nested PCR; 1E–7E, name of each epimastigote clonal population; PE, epimastigote forms of the parental cell line; PT, trypomastigote forms of the parental cell line.
    Figure Legend Snippet: Analysis of MASP clonal expression by capillary electrophoresis and western blot. ( a ) In silico gel obtained by PCR fingerprinting analysing PCR products of three-step nested PCR for each isogenic line. On the top, a dendogram based on the similarity of the MASP amplicons pattern. The distances are measured at the right side of the dendogram with the percentage of similarity among the samples. ( b ) Bar graph representing the number of MASP amplicons obtained in the third step of the nested PCR for each clone and the parental strain. ( c ) Number of amplicons obtained from the second and the third steps of the nested PCR in the trypomastigotes of the parental cell line. ( d ) Frequency of bands according to their size amplified from the third nested PCR of the epimastigote stage for all the clones and from the epimastigote and trypomastigote forms of the parental cell line. ( e ) Western blot analysis of total protein extracts from the epimastigote forms of all the clones, and the epimastigote and trypomastigote stage of the parental strain using anti-MASP-Ag mouse antisera. Anti-tubulin sera were used as loading control and pre-immune sera as negative control. Below, bar graphs representing the ratio 45 kDa/tubulin and 40 kDa/tubulin. Second PCR and third PCR are the products of the second and third PCRs of the three-step nested PCR; 1E–7E, name of each epimastigote clonal population; PE, epimastigote forms of the parental cell line; PT, trypomastigote forms of the parental cell line.

    Techniques Used: Expressing, Electrophoresis, Western Blot, In Silico, Polymerase Chain Reaction, Nested PCR, Amplification, Clone Assay, Negative Control

    27) Product Images from "Heat-Shock Promoters: Targets for Evolution by P Transposable Elements in Drosophila"

    Article Title: Heat-Shock Promoters: Targets for Evolution by P Transposable Elements in Drosophila

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0020165

    Distinctive P Elements Revealed by Re-screening a Random Sample of P Element Insertion Sites in Natural Populations for Four Genes, Hsp23, Hsp27, Hsrω, and Hsp70 The P element insertion sites were selected from Gene Set I. A plus sign (+) indicates successful PCR amplification with one primer complementary to the focal gene and another complementary to a unique sequence in the P element (top), and thus reports the size and orientation of the P element; a minus sign (−) indicates no amplification. Table S7 provides sequences of these primers. At each insertion site in a population, one to six distinctive P elements segregated; these are designated a–f. For Hsp23, Hsp27, and Hsrω, nine insertion sites shared by two or more natural populations (indicated by boxes) and 17 unique insertion sites were re-screened. Amplicons that share a symbol (filled square [█], filled triangle [▴], filled circle •], etc.) occurred at the same integration site in different populations and were indistinguishable by size or orientation. For Hsp70, a five-copy gene in natural populations [ 17 ], the specific gene of insertion was not determined; thus, each distinctive amplicon (a–f) could represent insertion(s) at the same site in one to five of the Hsp70 genes. For population codes see Figure 1 . ORF, open reading frame.
    Figure Legend Snippet: Distinctive P Elements Revealed by Re-screening a Random Sample of P Element Insertion Sites in Natural Populations for Four Genes, Hsp23, Hsp27, Hsrω, and Hsp70 The P element insertion sites were selected from Gene Set I. A plus sign (+) indicates successful PCR amplification with one primer complementary to the focal gene and another complementary to a unique sequence in the P element (top), and thus reports the size and orientation of the P element; a minus sign (−) indicates no amplification. Table S7 provides sequences of these primers. At each insertion site in a population, one to six distinctive P elements segregated; these are designated a–f. For Hsp23, Hsp27, and Hsrω, nine insertion sites shared by two or more natural populations (indicated by boxes) and 17 unique insertion sites were re-screened. Amplicons that share a symbol (filled square [█], filled triangle [▴], filled circle •], etc.) occurred at the same integration site in different populations and were indistinguishable by size or orientation. For Hsp70, a five-copy gene in natural populations [ 17 ], the specific gene of insertion was not determined; thus, each distinctive amplicon (a–f) could represent insertion(s) at the same site in one to five of the Hsp70 genes. For population codes see Figure 1 . ORF, open reading frame.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    28) Product Images from "Genome-Wide DNA Methylation and Gene Expression Analyses of Monozygotic Twins Discordant for Intelligence Levels"

    Article Title: Genome-Wide DNA Methylation and Gene Expression Analyses of Monozygotic Twins Discordant for Intelligence Levels

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047081

    DNA methylation status of the 5′-regions of ARHGAP18 and OR4D10 analyzed by bisulfite sequencing along with the quantitative RT-PCR analysis for ARHGAP18 expression. A, B. DNA methylation status analyzed by bisulfite sequencing. Schematic representation (top) for the relative position of CpGs within amplified regions and methylation profiling by bisulfite sequencing (bottom). The numbers at the ends of amplicons indicate the genome coordinates relative to the NCBI Build 36 genome assembly. Of all 27 loci identified by screening for epigenetically regulated candidate genes, A. ARHGAP18 and B. OR4D10 were validated by direct bisulfite sequencing. At least 30 clones were sequenced for each locus. Open squares indicate unmethylated CpG nucleotides and closed squares indicate methylated ones. Rows indicate the methylation status of each colony sequenced, while columns indicate the positions of CpG nucleotides.The percentages below refer to the ratio of CpG methylation. C. qRT-PCR analysis of ARHGAP18 mRNA relative to GAPDH in the twin pair ID 9. ARHGAP18 expression in twin 9A untreated with 5-azadC was normalized to 1. Error bars indicate ± SD. P -value was calculated using Mann-Whitney U test with asterisks indicating statistical significance ( p
    Figure Legend Snippet: DNA methylation status of the 5′-regions of ARHGAP18 and OR4D10 analyzed by bisulfite sequencing along with the quantitative RT-PCR analysis for ARHGAP18 expression. A, B. DNA methylation status analyzed by bisulfite sequencing. Schematic representation (top) for the relative position of CpGs within amplified regions and methylation profiling by bisulfite sequencing (bottom). The numbers at the ends of amplicons indicate the genome coordinates relative to the NCBI Build 36 genome assembly. Of all 27 loci identified by screening for epigenetically regulated candidate genes, A. ARHGAP18 and B. OR4D10 were validated by direct bisulfite sequencing. At least 30 clones were sequenced for each locus. Open squares indicate unmethylated CpG nucleotides and closed squares indicate methylated ones. Rows indicate the methylation status of each colony sequenced, while columns indicate the positions of CpG nucleotides.The percentages below refer to the ratio of CpG methylation. C. qRT-PCR analysis of ARHGAP18 mRNA relative to GAPDH in the twin pair ID 9. ARHGAP18 expression in twin 9A untreated with 5-azadC was normalized to 1. Error bars indicate ± SD. P -value was calculated using Mann-Whitney U test with asterisks indicating statistical significance ( p

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, Quantitative RT-PCR, Expressing, Amplification, Methylation, Clone Assay, CpG Methylation Assay, MANN-WHITNEY

    29) Product Images from "Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C InfectionThe Cat and Mouse of HIV-1 Antibody EscapeEscape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways"

    Article Title: Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C InfectionThe Cat and Mouse of HIV-1 Antibody EscapeEscape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000598

    Amino acid alignment of the V1V2 (green) and C3 (blue) regions of single genome amplicons of CAP88. Amplicons were derived from 1 month p.i. (yellow bar), 6 months p.i. (red bar) and 12 month p.i. (blue bars). Amplicons highlighted in bold text were cloned for neutralization assays. Potential N-linked glycosylation sites are highlighted in gray, dashes indicate deletions.
    Figure Legend Snippet: Amino acid alignment of the V1V2 (green) and C3 (blue) regions of single genome amplicons of CAP88. Amplicons were derived from 1 month p.i. (yellow bar), 6 months p.i. (red bar) and 12 month p.i. (blue bars). Amplicons highlighted in bold text were cloned for neutralization assays. Potential N-linked glycosylation sites are highlighted in gray, dashes indicate deletions.

    Techniques Used: Derivative Assay, Clone Assay, Neutralization

    30) Product Images from "Accuracy estimation of foamy virus genome copying"

    Article Title: Accuracy estimation of foamy virus genome copying

    Journal: Retrovirology

    doi: 10.1186/1742-4690-6-32

    Template Switching (TS) rate of foamy viral replication . (A) MD9-derived PFV vector viruses (KG81 and KG82) expressing hygromycin and neomycin resistance genes under control of a SFFV U3 promotor. CASI/II are cis -acting sequences required for FV vector transfer [ 71 ]. KG81 carries a point mutation in the hygromycin resistance gene that abolishes its function and destroys a SacII restriction site. KG812 carries a point mutation in the neomycin resistance gene that abolishes its function and destroys an EheII restriction site. The two mutations are 1 kb apart. (B) Distinction of TS events from superinfection with KG81 and KG82 by restriction pattern: amplification of the proviral sequences by PCR generates a 2.2 kb fragment with a SacI site at position 500 and an EheII site at position 1550. Amplicons of TS events carry the two intact restriction sites and show a restriction pattern depicted in the upper box, whereas amplicons of superinfected cells show the restriction pattern depicted in the lower box. (C) Representative digests of 10 clones from the TS experiment. All 10 clones show the expected pattern for TS events. Upper lane 3 and lower lane 2 show incomplete digests.
    Figure Legend Snippet: Template Switching (TS) rate of foamy viral replication . (A) MD9-derived PFV vector viruses (KG81 and KG82) expressing hygromycin and neomycin resistance genes under control of a SFFV U3 promotor. CASI/II are cis -acting sequences required for FV vector transfer [ 71 ]. KG81 carries a point mutation in the hygromycin resistance gene that abolishes its function and destroys a SacII restriction site. KG812 carries a point mutation in the neomycin resistance gene that abolishes its function and destroys an EheII restriction site. The two mutations are 1 kb apart. (B) Distinction of TS events from superinfection with KG81 and KG82 by restriction pattern: amplification of the proviral sequences by PCR generates a 2.2 kb fragment with a SacI site at position 500 and an EheII site at position 1550. Amplicons of TS events carry the two intact restriction sites and show a restriction pattern depicted in the upper box, whereas amplicons of superinfected cells show the restriction pattern depicted in the lower box. (C) Representative digests of 10 clones from the TS experiment. All 10 clones show the expected pattern for TS events. Upper lane 3 and lower lane 2 show incomplete digests.

    Techniques Used: Derivative Assay, Plasmid Preparation, Expressing, Mutagenesis, Amplification, Polymerase Chain Reaction, Clone Assay

    31) Product Images from "The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle"

    Article Title: The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv084

    MYH3 mRNA and embryonic myosin protein are expressed in both fetal and adult human skeletal muscle. ( A ) Ethidium bromide stained agarose gel: 100 bp ladder (Lane 1); amplicons of MYH3 (Lane 2) and MYH2 (Lane 3) from genomic DNA; amplicons of MYH3 (Lane
    Figure Legend Snippet: MYH3 mRNA and embryonic myosin protein are expressed in both fetal and adult human skeletal muscle. ( A ) Ethidium bromide stained agarose gel: 100 bp ladder (Lane 1); amplicons of MYH3 (Lane 2) and MYH2 (Lane 3) from genomic DNA; amplicons of MYH3 (Lane

    Techniques Used: Staining, Agarose Gel Electrophoresis

    32) Product Images from "Rapid Extraction from and Direct Identification in Clinical Samples of Methicillin-Resistant Staphylococci Using the PCR"

    Article Title: Rapid Extraction from and Direct Identification in Clinical Samples of Methicillin-Resistant Staphylococci Using the PCR

    Journal: Journal of Clinical Microbiology

    doi:

    Agarose gel electrophoresis of genomic DNA extracted by different methods from S. aureus 450M, amplified with MRSA primer sets. Lane 1, 100-bp DNA ladder (100 to 15,000 bp and 2,072-bp fragment) (Life Technologies); lane 2, DNA PCR control from RTG PCR beads (Pharmacia Biotech) (500 bp); lanes 3 to 5, QIAGEN method-extracted DNA bacterial culture; lanes 6 to 8, BB+C-extracted DNA; lanes 9 to 11, previously made amplicons (positive control) generated from the mecA gene (997 bp); S. aureus 16S rRNA gene (750 bp), and bacterial 16S rRNA gene (292 bp); lanes 12 to 14, no DNA template (negative control) with each primer set. Lanes 3 and 6 contain amplicons PCR generated with the mecA gene primer set, lanes 4 and 7 contain amplicons PCR generated with the S. aureus 16S rRNA gene primer set, and lanes 5 and 8 contain amplicons PCR generated with the bacterial 16S rRNA gene primer set.
    Figure Legend Snippet: Agarose gel electrophoresis of genomic DNA extracted by different methods from S. aureus 450M, amplified with MRSA primer sets. Lane 1, 100-bp DNA ladder (100 to 15,000 bp and 2,072-bp fragment) (Life Technologies); lane 2, DNA PCR control from RTG PCR beads (Pharmacia Biotech) (500 bp); lanes 3 to 5, QIAGEN method-extracted DNA bacterial culture; lanes 6 to 8, BB+C-extracted DNA; lanes 9 to 11, previously made amplicons (positive control) generated from the mecA gene (997 bp); S. aureus 16S rRNA gene (750 bp), and bacterial 16S rRNA gene (292 bp); lanes 12 to 14, no DNA template (negative control) with each primer set. Lanes 3 and 6 contain amplicons PCR generated with the mecA gene primer set, lanes 4 and 7 contain amplicons PCR generated with the S. aureus 16S rRNA gene primer set, and lanes 5 and 8 contain amplicons PCR generated with the bacterial 16S rRNA gene primer set.

    Techniques Used: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Positive Control, Generated, Negative Control

    33) Product Images from "Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development"

    Article Title: Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gng095

    PCR confirmation of insertion of the transposable bs r cassette in the qkgA locus and determination of the transposon insertion point. ( A ) A schematic diagram of the qkgA locus and the inserted bs r cassette. The orange line indicates that part of the qkgA open reading frame which was used as the target for disruption. The position of insertion of the bs r cassette is marked with a black triangle. The two primers used for PCR confirmation are indicated with arrows and marked primers 1 and 2. ( B ) Two examples of clones. Genomic DNA was isolated from the clones grown in selective medium containing blasticidin. Lanes 1 and 2 show the results of PCR with the primers described in (A). The sample analysed in lane 1 shows successful insertion of the selection cassette into qkgA , while the absence of a band in lane 2 suggests a non-homologous insertion of the cassette elsewhere in the chromosome. ( C ) Following PCR confirmation of transposon insertion in the genomic target, the insertion point was estimated from the size of the amplicons. The arrows indicate the approximate insertion points of two separate clones, QkgA-1 and QkgA-2.
    Figure Legend Snippet: PCR confirmation of insertion of the transposable bs r cassette in the qkgA locus and determination of the transposon insertion point. ( A ) A schematic diagram of the qkgA locus and the inserted bs r cassette. The orange line indicates that part of the qkgA open reading frame which was used as the target for disruption. The position of insertion of the bs r cassette is marked with a black triangle. The two primers used for PCR confirmation are indicated with arrows and marked primers 1 and 2. ( B ) Two examples of clones. Genomic DNA was isolated from the clones grown in selective medium containing blasticidin. Lanes 1 and 2 show the results of PCR with the primers described in (A). The sample analysed in lane 1 shows successful insertion of the selection cassette into qkgA , while the absence of a band in lane 2 suggests a non-homologous insertion of the cassette elsewhere in the chromosome. ( C ) Following PCR confirmation of transposon insertion in the genomic target, the insertion point was estimated from the size of the amplicons. The arrows indicate the approximate insertion points of two separate clones, QkgA-1 and QkgA-2.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Isolation, Selection

    34) Product Images from "Rapid and Accurate Identification of Human Isolates of Pasteurella and Related Species by Sequencing the sodA Gene"

    Article Title: Rapid and Accurate Identification of Human Isolates of Pasteurella and Related Species by Sequencing the sodA Gene

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.43.5.2307-2314.2005

    Amplification of type strains of Pasteurella and related species with the primers d1 and d2 and separation of the sodA int amplicons (arrowheads) by 1% agarose gel electrophoresis. Lanes: 1 and 21, 50-bp DNA ladder (Fermentas, Lithuania); 2, P. avium ; 3, P. gallinarum ; 4, P. volantium ; 5, P. skyensis ; 6, P. testudinis ; 7, A. ureae ; 8, Mannheimia sp.; 9, P. trehalosi ; 10, P. canis ; 11, P. dagmatis ; 12, P. multocida ; 13, P. stomatis ; 14, P. aerogenes ; 15, P. mairii ; 16, G. anatis ; 17, H. parainfluenzae ; 18, P. bettyae ; 19, P. langaaensis , 20, P. caballi. The size of the amplicons is either 503 bp (lanes 2 to 9), 506 bp (lanes 10 to 17), 515 bp (lane 18), 518 bp (lane 19), or 527 bp (lane 20).
    Figure Legend Snippet: Amplification of type strains of Pasteurella and related species with the primers d1 and d2 and separation of the sodA int amplicons (arrowheads) by 1% agarose gel electrophoresis. Lanes: 1 and 21, 50-bp DNA ladder (Fermentas, Lithuania); 2, P. avium ; 3, P. gallinarum ; 4, P. volantium ; 5, P. skyensis ; 6, P. testudinis ; 7, A. ureae ; 8, Mannheimia sp.; 9, P. trehalosi ; 10, P. canis ; 11, P. dagmatis ; 12, P. multocida ; 13, P. stomatis ; 14, P. aerogenes ; 15, P. mairii ; 16, G. anatis ; 17, H. parainfluenzae ; 18, P. bettyae ; 19, P. langaaensis , 20, P. caballi. The size of the amplicons is either 503 bp (lanes 2 to 9), 506 bp (lanes 10 to 17), 515 bp (lane 18), 518 bp (lane 19), or 527 bp (lane 20).

    Techniques Used: Amplification, Agarose Gel Electrophoresis

    35) Product Images from "Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils"

    Article Title: Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02833-16

    Sybr Green- and TaqMan-based qPCR quantification of amplicons from representative pure cultures or endophytes of Frankia clusters 1, 2, 3, and 4 and of subgroups a, b, and c within cluster 1. Individual amplicons or mixtures of amplicons were quantified
    Figure Legend Snippet: Sybr Green- and TaqMan-based qPCR quantification of amplicons from representative pure cultures or endophytes of Frankia clusters 1, 2, 3, and 4 and of subgroups a, b, and c within cluster 1. Individual amplicons or mixtures of amplicons were quantified

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction

    36) Product Images from "The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs"

    Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagrm.2013.09.007

    The ACE2 gene promoter is bipartite in humans and mice. (A) Diagram of the human ACE2 promoter with two conserved regions separated by a repetitive Alu element giving rise to two transcript forms encoding the same protein. (B) Design of DPT and PPT assays involves a common reverse primer, unique forward primers, and unique Taqman probes. (C) The DPT and PPT assays were run with different concentration of pCRII-TOPO plasmids containing cloned DPT and PPT amplicons as samples. A water sample was run as a no-template control. The cycle-threshold values (Ct) were recorded. (D) Comparisons of the concentrations of DPT and PPT in mouse tissues. Concentrations are shown as the C t values obtained for RNA samples diluted to 20 ng/μl. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. The lung, kidney, heart, and brain were harvested from 3 female mice and the pancreas from 5 male mice. *: p
    Figure Legend Snippet: The ACE2 gene promoter is bipartite in humans and mice. (A) Diagram of the human ACE2 promoter with two conserved regions separated by a repetitive Alu element giving rise to two transcript forms encoding the same protein. (B) Design of DPT and PPT assays involves a common reverse primer, unique forward primers, and unique Taqman probes. (C) The DPT and PPT assays were run with different concentration of pCRII-TOPO plasmids containing cloned DPT and PPT amplicons as samples. A water sample was run as a no-template control. The cycle-threshold values (Ct) were recorded. (D) Comparisons of the concentrations of DPT and PPT in mouse tissues. Concentrations are shown as the C t values obtained for RNA samples diluted to 20 ng/μl. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. The lung, kidney, heart, and brain were harvested from 3 female mice and the pancreas from 5 male mice. *: p

    Techniques Used: Mouse Assay, Concentration Assay, Clone Assay, Amplification

    37) Product Images from "Mutant Enrichment with 3?-Modified Oligonucleotides"

    Article Title: Mutant Enrichment with 3?-Modified Oligonucleotides

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.07.003

    Potential applicability of MEMO to quantitative real-time PCR and/or subsequent melting curve analysis. A: An example of serially diluted specimens with EGFR T790M mutations detected through a real-time PCR assay using a DNA-intercalating fluorescence dye illustrates different fluorescence curves according to the mutant allele concentration. B: The standard curves generated in quadruplicate runs have a very linear correlation ( r 2 = 0.991) in dilutions ranging from 1.0 × 10 0 to 1.0 × 10 −3 (PCR efficiency: 1.45). C: Subsequent high-resolution melting curve analysis demonstrates a higher T m (84.3°C to 84.4°C) in dilutions with a high mutant allele concentration (1.0 × 10 0 , 1.0 × 10 −1 , and 1.0 × 10 −2 ) compared to that of normal samples (83.7°C), whereas samples with low mutant allele concentrations (1.0 × 10 −3 ) produce heterozygous melting curves. D: Amplicons were sequenced, and the results were in concordance with those of the high-resolution melting curve analysis (ie, homozygous mutant peaks in samples with a high mutant allele concentration and heterozygous peaks in those with a low mutant allele concentration). The target mutation site is indicated with an arrow .
    Figure Legend Snippet: Potential applicability of MEMO to quantitative real-time PCR and/or subsequent melting curve analysis. A: An example of serially diluted specimens with EGFR T790M mutations detected through a real-time PCR assay using a DNA-intercalating fluorescence dye illustrates different fluorescence curves according to the mutant allele concentration. B: The standard curves generated in quadruplicate runs have a very linear correlation ( r 2 = 0.991) in dilutions ranging from 1.0 × 10 0 to 1.0 × 10 −3 (PCR efficiency: 1.45). C: Subsequent high-resolution melting curve analysis demonstrates a higher T m (84.3°C to 84.4°C) in dilutions with a high mutant allele concentration (1.0 × 10 0 , 1.0 × 10 −1 , and 1.0 × 10 −2 ) compared to that of normal samples (83.7°C), whereas samples with low mutant allele concentrations (1.0 × 10 −3 ) produce heterozygous melting curves. D: Amplicons were sequenced, and the results were in concordance with those of the high-resolution melting curve analysis (ie, homozygous mutant peaks in samples with a high mutant allele concentration and heterozygous peaks in those with a low mutant allele concentration). The target mutation site is indicated with an arrow .

    Techniques Used: Real-time Polymerase Chain Reaction, Fluorescence, Mutagenesis, Concentration Assay, Generated, Polymerase Chain Reaction

    38) Product Images from "Infectious Molecular Clone of a Recently Transmitted Pediatric Human Immunodeficiency Virus Clade C Isolate from Africa: Evidence of Intraclade Recombination"

    Article Title: Infectious Molecular Clone of a Recently Transmitted Pediatric Human Immunodeficiency Virus Clade C Isolate from Africa: Evidence of Intraclade Recombination

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.24.14066-14069.2004

    Strategy for the cloning of HIV1084i. Full-length HIV1084i was constructed from two subgenomic amplicons containing NotI and AscI restriction sites at alternate ends of the molecule. NotI restriction sites were added to the LTR primers (1 and 4), while
    Figure Legend Snippet: Strategy for the cloning of HIV1084i. Full-length HIV1084i was constructed from two subgenomic amplicons containing NotI and AscI restriction sites at alternate ends of the molecule. NotI restriction sites were added to the LTR primers (1 and 4), while

    Techniques Used: Clone Assay, Construct

    39) Product Images from "Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells"

    Article Title: Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2014.138

    Transcription of CR2/CD21 proceeds from a single predominant start site in the majority of mature B cells. ( a ) 5′RACE generates a single major PCR product in Ramos (lane 3) and Raji (lane 6), while no PCR amplicons are detected in Reh, K562 or
    Figure Legend Snippet: Transcription of CR2/CD21 proceeds from a single predominant start site in the majority of mature B cells. ( a ) 5′RACE generates a single major PCR product in Ramos (lane 3) and Raji (lane 6), while no PCR amplicons are detected in Reh, K562 or

    Techniques Used: Polymerase Chain Reaction

    40) Product Images from "Identification and Expression of Benzylsuccinate Synthase Genes in a Toluene-Degrading Methanogenic Consortium ▿"

    Article Title: Identification and Expression of Benzylsuccinate Synthase Genes in a Toluene-Degrading Methanogenic Consortium ▿

    Journal:

    doi: 10.1128/AEM.01904-06

    Agarose gels showing amplicons from RT-PCRs performed using cultures amended with toluene (T) or benzoate (B). Amplification was carried out using specific bssA -2 primers (panel A) and Eub-1 (16S rRNA gene) primers (panel B) listed in Table
    Figure Legend Snippet: Agarose gels showing amplicons from RT-PCRs performed using cultures amended with toluene (T) or benzoate (B). Amplification was carried out using specific bssA -2 primers (panel A) and Eub-1 (16S rRNA gene) primers (panel B) listed in Table

    Techniques Used: Amplification

    Related Articles

    Clone Assay:

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
    Article Snippet: .. Amplicons from AhPLD1 , AhPLD2 , and AhLOX7 were cloned with Zero-Blunt PCR cloning kit (Life Technologies, catalog no. K 2700–20) and colonies were sent to the Georgia Genomics Facility for Sanger sequencing. .. Sequences were analysed by Geneious software ( http://www.geneious.com/ ).

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
    Article Snippet: .. Mutants identified from CAPS assay and SSCP were further confirmed by Sanger sequencing of amplicons (single copy genes) or performing PCR cloning with Zero-Blunt PCR cloning kit (Life Technologies, catalogue no. K 2700–20) prior to sequencing. .. The mutation effect was analysed by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/ ) with default parameters [ ].

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius
    Article Snippet: .. Cloning of Ct PAL and Ct CHS amplicons into sequencing vector To clone the amplicons of Ct PAL and Ct CHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited. .. In brief, based upon blue/white screening, recombinant colonies were selected for DNA extraction by GF-1 Plasmid DNA Extraction Kit (Vivantis).

    Agarose Gel Electrophoresis:

    Article Title: Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as Vectors of Leishmania infantum in Tbilisi, Georgia
    Article Snippet: .. Amplicons were produced in a GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad, CA) thermocycler and the products visualized on cyanine-stained 1.5% agarose gel alongside a TrackIt 100 bp DNA ladder (Invitrogen, Carlsbad, CA). .. Positive control DNA templates were extracted from Phlebotomus perniciosus fed artificially through a membrane on dog blood and Phlebotomus duboscqi fed on a human and were processed in a manner identical to field-caught specimens.

    In Vitro:

    Article Title: Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer
    Article Snippet: .. Purified amplicons were in-vitro transcribed to synthesize dsRNA using MEGAscript T7 kit (Ambion, Austin, TX). .. Amplicon from Escherichia coli malE gene was used to prepare control dsRNA. dsRNA injections were performed on the first day of final instar larvae, 0 h pupa or newly emerged male and female adults (~6 h PAE).

    Synthesized:

    Article Title: Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR(R)Green Real-Time PCR Methods and High Resolution Melting Analysis
    Article Snippet: .. Plasmids harbouring the corresponding amplicons for the VTEC PCR methods were synthesized at GeneArt as previously described . .. Primer pair specificity for each target was assessed by testing amplification of genomic DNA of target-containing and target-lacking strains ( ).

    Purification:

    Article Title: Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer
    Article Snippet: .. Purified amplicons were in-vitro transcribed to synthesize dsRNA using MEGAscript T7 kit (Ambion, Austin, TX). .. Amplicon from Escherichia coli malE gene was used to prepare control dsRNA. dsRNA injections were performed on the first day of final instar larvae, 0 h pupa or newly emerged male and female adults (~6 h PAE).

    SYBR Green Assay:

    Article Title: Many LINE1 elements contribute to the transcriptome of human somatic cells
    Article Snippet: .. The SYBR green method was used for most amplicons (SYBR Green PCR master mix (2×); Applied Biosystems), in conjunction with oligonucleotide pairs F/R as indicated (Figures , , and ; Additional data file 5). .. The exception was the L1 at 4p15.32, which was assayed with the TaqMan method (TaqMan Gene Expression master mix, Applied Biosystems) using the oligonucleotide pair 4p15.32R/L1HsSP1A and the antisense strand 4p15.32 TaqMan probe (Additional data file 5).

    Polymerase Chain Reaction:

    Article Title: Many LINE1 elements contribute to the transcriptome of human somatic cells
    Article Snippet: .. The SYBR green method was used for most amplicons (SYBR Green PCR master mix (2×); Applied Biosystems), in conjunction with oligonucleotide pairs F/R as indicated (Figures , , and ; Additional data file 5). .. The exception was the L1 at 4p15.32, which was assayed with the TaqMan method (TaqMan Gene Expression master mix, Applied Biosystems) using the oligonucleotide pair 4p15.32R/L1HsSP1A and the antisense strand 4p15.32 TaqMan probe (Additional data file 5).

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
    Article Snippet: .. Amplicons from AhPLD1 , AhPLD2 , and AhLOX7 were cloned with Zero-Blunt PCR cloning kit (Life Technologies, catalog no. K 2700–20) and colonies were sent to the Georgia Genomics Facility for Sanger sequencing. .. Sequences were analysed by Geneious software ( http://www.geneious.com/ ).

    Article Title: Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR(R)Green Real-Time PCR Methods and High Resolution Melting Analysis
    Article Snippet: .. Plasmids harbouring the corresponding amplicons for the VTEC PCR methods were synthesized at GeneArt as previously described . .. Primer pair specificity for each target was assessed by testing amplification of genomic DNA of target-containing and target-lacking strains ( ).

    Article Title: Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as Vectors of Leishmania infantum in Tbilisi, Georgia
    Article Snippet: .. Amplicons were produced in a GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad, CA) thermocycler and the products visualized on cyanine-stained 1.5% agarose gel alongside a TrackIt 100 bp DNA ladder (Invitrogen, Carlsbad, CA). .. Positive control DNA templates were extracted from Phlebotomus perniciosus fed artificially through a membrane on dog blood and Phlebotomus duboscqi fed on a human and were processed in a manner identical to field-caught specimens.

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
    Article Snippet: .. Mutants identified from CAPS assay and SSCP were further confirmed by Sanger sequencing of amplicons (single copy genes) or performing PCR cloning with Zero-Blunt PCR cloning kit (Life Technologies, catalogue no. K 2700–20) prior to sequencing. .. The mutation effect was analysed by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/ ) with default parameters [ ].

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius
    Article Snippet: .. Cloning of Ct PAL and Ct CHS amplicons into sequencing vector To clone the amplicons of Ct PAL and Ct CHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited. .. In brief, based upon blue/white screening, recombinant colonies were selected for DNA extraction by GF-1 Plasmid DNA Extraction Kit (Vivantis).

    Sequencing:

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
    Article Snippet: .. Amplicons from AhPLD1 , AhPLD2 , and AhLOX7 were cloned with Zero-Blunt PCR cloning kit (Life Technologies, catalog no. K 2700–20) and colonies were sent to the Georgia Genomics Facility for Sanger sequencing. .. Sequences were analysed by Geneious software ( http://www.geneious.com/ ).

    Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
    Article Snippet: .. Mutants identified from CAPS assay and SSCP were further confirmed by Sanger sequencing of amplicons (single copy genes) or performing PCR cloning with Zero-Blunt PCR cloning kit (Life Technologies, catalogue no. K 2700–20) prior to sequencing. .. The mutation effect was analysed by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/ ) with default parameters [ ].

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius
    Article Snippet: .. Cloning of Ct PAL and Ct CHS amplicons into sequencing vector To clone the amplicons of Ct PAL and Ct CHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited. .. In brief, based upon blue/white screening, recombinant colonies were selected for DNA extraction by GF-1 Plasmid DNA Extraction Kit (Vivantis).

    Article Title: Simultaneous Detection of Major Pome Fruit Viruses and a Viroid
    Article Snippet: .. Amplicons were sequenced using an ABI Prism 310 with an ABI prism Big Dye-Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). ..

    Produced:

    Article Title: Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as Vectors of Leishmania infantum in Tbilisi, Georgia
    Article Snippet: .. Amplicons were produced in a GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad, CA) thermocycler and the products visualized on cyanine-stained 1.5% agarose gel alongside a TrackIt 100 bp DNA ladder (Invitrogen, Carlsbad, CA). .. Positive control DNA templates were extracted from Phlebotomus perniciosus fed artificially through a membrane on dog blood and Phlebotomus duboscqi fed on a human and were processed in a manner identical to field-caught specimens.

    Plasmid Preparation:

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius
    Article Snippet: .. Cloning of Ct PAL and Ct CHS amplicons into sequencing vector To clone the amplicons of Ct PAL and Ct CHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited. .. In brief, based upon blue/white screening, recombinant colonies were selected for DNA extraction by GF-1 Plasmid DNA Extraction Kit (Vivantis).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Thermo Fisher pcr amplicon
    CRISPR/Cas9-mediated deletion mutation of MtGA2ox10 . ( A ) Gene structure of MtGA2ox10 . Target sequences of two guide RNAs, G851 and G907, were designed on exon 3. Relative nucleotide positions of the PAM sites marked in boxes are numbered from the start codon. Restriction sites for BsrD I and Eco105 I are also presented. ( B ) Restriction maps of the wild type <t>PCR</t> products amplified with the 2347-F and 2905-R primers for genotyping by restriction fragment length polymorphism (RFLP), using BsrD I (left) or Eco105 I (right). ( C ) T-DNA structure of the Cas9 binary construct G851.907 for the deletion mutation of MtGA2ox10 . Two single guide RNAs (sgRNAs) (G907 and G851 under MtU6–8 promoters) were tandem-assembled into the binary pGK3304 vector, which contains the GFP::BAR selection marker and Cas9::NLS under CaMV 35S promoters. ( D ) PCR-RFLP genotyping of A. rhizogenes -transformed roots harboring G851.907. PCR amplicons from four root samples of the pGK3304 empty vector (NULL) and seven root samples of G851.907 (G851.907) were digested independently by BsrD I (left lane) or Eco105 I (right lane). Note that all the amplicons from the NULL samples were digested to fragments of the expected sizes, as shown in the left margin of the agarose gel. In contrast, none of the amplicons from G851.907 samples were digested, indicating disruption of the restriction sites for BsrD I and Eco105 I. Sample 6 shows an increased <t>amplicon</t> size, presumably due to an insertion. ( E ) Sanger sequencing chromatograms for the PCR amplicons of the G851.907 KO-1, -2, and -3. The PAM sequence for G851 is denoted in the box and the expected cleavage site (−4 bp from PAM) is marked with an arrow, where mixed peaks appear in the sequencing chromatograms. ( F ) Sequences of each allele in the G851.907 KO-1, -2 and -3. The PAM sequences for sgRNAs are shown in boxes. Predicted changes in the protein structure by deletion mutations are described on the right.
    Pcr Amplicon, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr amplicon/product/Thermo Fisher
    Average 93 stars, based on 77 article reviews
    Price from $9.99 to $1999.99
    pcr amplicon - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    95
    Thermo Fisher amplicons
    Expression patterns of Ct PAL and Ct CHS genes after wounding (A) and during salinity stress (B) Samplings were carried out at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of <t>amplicons:</t> Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.
    Amplicons, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2047 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplicons/product/Thermo Fisher
    Average 95 stars, based on 2047 article reviews
    Price from $9.99 to $1999.99
    amplicons - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    88
    Thermo Fisher amplicon sequencing libraries
    Primer dimer formation in tailed <t>amplicon</t> method. A) Agilent TapeStation trace for a library prepared from samples with N1 and N2 Ct values between ~20-40 using the tailed amplicon v1 (2 pool amplification) workflow. B) Mean read 1 quality score for samples prepared with the tailed amplicon v1 (2 pool amplification) workflow amplified for either 25 or 35 PCR cycles. C) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v1 (2 pool amplification) workflow amplified for either 25 or 35 PCR cycles. D) Agilent Bioanalyzer trace for a library prepared from samples with N1 and N2 Ct values between ~20-35 using the tailed amplicon v2 (4 pool amplification) workflow. E) Mean read 1 quality score for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. F) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. G) 2% agarose gel showing the presence of primer dimers particularly in high N1/N2 Ct samples when indexed using different PCR cycling conditions. Arrow indicates primer dimers on gel.
    Amplicon Sequencing Libraries, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplicon sequencing libraries/product/Thermo Fisher
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    amplicon sequencing libraries - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    CRISPR/Cas9-mediated deletion mutation of MtGA2ox10 . ( A ) Gene structure of MtGA2ox10 . Target sequences of two guide RNAs, G851 and G907, were designed on exon 3. Relative nucleotide positions of the PAM sites marked in boxes are numbered from the start codon. Restriction sites for BsrD I and Eco105 I are also presented. ( B ) Restriction maps of the wild type PCR products amplified with the 2347-F and 2905-R primers for genotyping by restriction fragment length polymorphism (RFLP), using BsrD I (left) or Eco105 I (right). ( C ) T-DNA structure of the Cas9 binary construct G851.907 for the deletion mutation of MtGA2ox10 . Two single guide RNAs (sgRNAs) (G907 and G851 under MtU6–8 promoters) were tandem-assembled into the binary pGK3304 vector, which contains the GFP::BAR selection marker and Cas9::NLS under CaMV 35S promoters. ( D ) PCR-RFLP genotyping of A. rhizogenes -transformed roots harboring G851.907. PCR amplicons from four root samples of the pGK3304 empty vector (NULL) and seven root samples of G851.907 (G851.907) were digested independently by BsrD I (left lane) or Eco105 I (right lane). Note that all the amplicons from the NULL samples were digested to fragments of the expected sizes, as shown in the left margin of the agarose gel. In contrast, none of the amplicons from G851.907 samples were digested, indicating disruption of the restriction sites for BsrD I and Eco105 I. Sample 6 shows an increased amplicon size, presumably due to an insertion. ( E ) Sanger sequencing chromatograms for the PCR amplicons of the G851.907 KO-1, -2, and -3. The PAM sequence for G851 is denoted in the box and the expected cleavage site (−4 bp from PAM) is marked with an arrow, where mixed peaks appear in the sequencing chromatograms. ( F ) Sequences of each allele in the G851.907 KO-1, -2 and -3. The PAM sequences for sgRNAs are shown in boxes. Predicted changes in the protein structure by deletion mutations are described on the right.

    Journal: Scientific Reports

    Article Title: MtGA2ox10 encoding C20-GA2-oxidase regulates rhizobial infection and nodule development in Medicago truncatula

    doi: 10.1038/s41598-019-42407-3

    Figure Lengend Snippet: CRISPR/Cas9-mediated deletion mutation of MtGA2ox10 . ( A ) Gene structure of MtGA2ox10 . Target sequences of two guide RNAs, G851 and G907, were designed on exon 3. Relative nucleotide positions of the PAM sites marked in boxes are numbered from the start codon. Restriction sites for BsrD I and Eco105 I are also presented. ( B ) Restriction maps of the wild type PCR products amplified with the 2347-F and 2905-R primers for genotyping by restriction fragment length polymorphism (RFLP), using BsrD I (left) or Eco105 I (right). ( C ) T-DNA structure of the Cas9 binary construct G851.907 for the deletion mutation of MtGA2ox10 . Two single guide RNAs (sgRNAs) (G907 and G851 under MtU6–8 promoters) were tandem-assembled into the binary pGK3304 vector, which contains the GFP::BAR selection marker and Cas9::NLS under CaMV 35S promoters. ( D ) PCR-RFLP genotyping of A. rhizogenes -transformed roots harboring G851.907. PCR amplicons from four root samples of the pGK3304 empty vector (NULL) and seven root samples of G851.907 (G851.907) were digested independently by BsrD I (left lane) or Eco105 I (right lane). Note that all the amplicons from the NULL samples were digested to fragments of the expected sizes, as shown in the left margin of the agarose gel. In contrast, none of the amplicons from G851.907 samples were digested, indicating disruption of the restriction sites for BsrD I and Eco105 I. Sample 6 shows an increased amplicon size, presumably due to an insertion. ( E ) Sanger sequencing chromatograms for the PCR amplicons of the G851.907 KO-1, -2, and -3. The PAM sequence for G851 is denoted in the box and the expected cleavage site (−4 bp from PAM) is marked with an arrow, where mixed peaks appear in the sequencing chromatograms. ( F ) Sequences of each allele in the G851.907 KO-1, -2 and -3. The PAM sequences for sgRNAs are shown in boxes. Predicted changes in the protein structure by deletion mutations are described on the right.

    Article Snippet: The resulting PCR amplicon was purified by agarose gel electrophoresis and cloned into pDONR221 using BP Clonase II (Thermo Fisher Scientific).

    Techniques: CRISPR, Mutagenesis, Polymerase Chain Reaction, Amplification, Construct, Plasmid Preparation, Selection, Marker, Transformation Assay, Agarose Gel Electrophoresis, Sequencing

    Expression patterns of Ct PAL and Ct CHS genes after wounding (A) and during salinity stress (B) Samplings were carried out at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.

    Journal: Bioscience Reports

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    doi: 10.1042/BSR20140026

    Figure Lengend Snippet: Expression patterns of Ct PAL and Ct CHS genes after wounding (A) and during salinity stress (B) Samplings were carried out at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.

    Article Snippet: Cloning of Ct PAL and Ct CHS amplicons into sequencing vector To clone the amplicons of Ct PAL and Ct CHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited.

    Techniques: Expressing, HAT Assay, Polymerase Chain Reaction

    Expression patterns of Ct PAL and Ct CHS genes after SA treatment with 0.1 mM (A) and 1 mM (B) concentrations Samplings were done at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.

    Journal: Bioscience Reports

    Article Title: Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    doi: 10.1042/BSR20140026

    Figure Lengend Snippet: Expression patterns of Ct PAL and Ct CHS genes after SA treatment with 0.1 mM (A) and 1 mM (B) concentrations Samplings were done at 0, 3, 6, 12, 24 and 48 hat. RNAs were extracted from all seedlings and treated with DNaseI. Subsequently, RNAs were reverse transcribed to corresponding cDNAs. Different PCR products intensities were referred to as temporal expression level of the genes. 18S rRNA transcription levels were considered as internal house-keeping gene control. Sizes of amplicons: Ct PAL: 267 bp; Ct CHS 559 bp; 18S rRNA: 199 bp.

    Article Snippet: Cloning of Ct PAL and Ct CHS amplicons into sequencing vector To clone the amplicons of Ct PAL and Ct CHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited.

    Techniques: Expressing, HAT Assay, Polymerase Chain Reaction

    Engineered cycloheximide resistance establishes genome editing conditions. ( A ) The design of a cycloheximide resistant allele, rpl36a P56Q , in S. rosetta . The protospacer adjacent motif (PAM, orange) next to the 56 th codon of rpl36a (Target, cyan), which is located on the second exon (thick black line labeled 2), provides a suitable site to design a gRNA that targets Sp Cas9 cleavage (sequence is shown underneath the locus schematic, and carets indicate the target cleavage site). A repair oligonucleotide (black line with knob) introduces a cycloheximide resistant allele, rpl36a P56Q (Mutation, purple), flanked by 100 bases of homologous sequence. The sequence of the edited allele is shown below. ( B–D ) A comparison of genotypes from populations of unedited cells ( B ), edited cells ( C ), and a strain established from a clonal isolate of edited cells ( D ) shows that cycloheximide selection enriches for rpl36a P56Q . The genotype for each population was determined by amplifying the locus with primers surrounding the editing site (black arrows in panel A) that did not overlap in sequence with the repair oligonucleotide. One of the primers had a T3 primer binding site for Sanger sequencing of amplicons (black arrow with flap). Remarkably, after selection, the wild-type allele was not detected ( B ). ( E ) S. rosetta uses repair oligonucleotides with > 20 nt homology arms for genome editing. Truncations of repair oligonucleotides encoding the rpl36a P56Q allele were designed in the same orientation as gRNAs (sense, black dots and lines) or the opposite orientation (antisense, gray dots and lines). 24 hr after S. rosetta recovered from transfections with repair templates and Sp Cas9 RNPs, cycloheximide was added to grow cells in selective media for five days, at which time the cells were harvested for counting cell density and for genotyping. Closed circles indicate that the consensus genotype of the cell population had the rpl36a P56Q allele in Sanger sequencing; whereas, open circles indicate that the cell population had the wild-type allele. E’ and E’’ show two independent trials. Notably, we observed a slight bias for repair oligonucleotides in the sense direction, particularly with shorter homology arms of 20-30 bases. Because repair templates in the sense orientation with 40–80 bases of homologous sequence resulted in the best editing, we performed subsequent optimization with a sense repair oligonucleotide that 50-base homology arms on each side of the double-stranded break. ( F ) Small quantities of Sp Cas9 RNPs are sufficient to initiate genome editing. Decreasing concentrations of Sp Cas9 RNP ( Sp Cas9 was the limiting factor) and a constant amount of repair template were transfected into S. rosetta . After characterizing genome editing outcomes by counting cell density and sequencing the consensus genotype (described in panel E), we found that low concentrations of Sp Cas9 (20 pmol) were sufficient to introduce the rpl36a P56Q allele. F’ and F’’ show two independent trials. ( G ) High concentrations of repair oligonucleotides increase genome editing efficiency. A serial dilution of a repair template was delivered into S. rosetta . The cell density and consensus genotypes from these experiments show that all concentrations of repair template can introduce the rpl36a P56Q allele, but the higher cell densities recovered after transfection with increasing concentrations of repair templates indicate more efficient editing. G’ and G’’ show two independent trials. ( H ) The addition of gRNAs stimulates genome editing. Genome editing was performed by delivering a repair oligonucleotide with Sp Cas9 without the addition of any gRNA or with a gRNA that was prepared from in vitro transcriptions (noted as gRNA in figure) or with a synthetic crRNA that was annealed to a synthetic tracrRNA (noted as crRNA). The consensus genotype and cell densities from these experiments show that gRNAs are necessary for editing and that gRNAs from either source were sufficient for editing. The dots show two independent experiments and lines show their average result.

    Journal: eLife

    Article Title: Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta

    doi: 10.7554/eLife.56193

    Figure Lengend Snippet: Engineered cycloheximide resistance establishes genome editing conditions. ( A ) The design of a cycloheximide resistant allele, rpl36a P56Q , in S. rosetta . The protospacer adjacent motif (PAM, orange) next to the 56 th codon of rpl36a (Target, cyan), which is located on the second exon (thick black line labeled 2), provides a suitable site to design a gRNA that targets Sp Cas9 cleavage (sequence is shown underneath the locus schematic, and carets indicate the target cleavage site). A repair oligonucleotide (black line with knob) introduces a cycloheximide resistant allele, rpl36a P56Q (Mutation, purple), flanked by 100 bases of homologous sequence. The sequence of the edited allele is shown below. ( B–D ) A comparison of genotypes from populations of unedited cells ( B ), edited cells ( C ), and a strain established from a clonal isolate of edited cells ( D ) shows that cycloheximide selection enriches for rpl36a P56Q . The genotype for each population was determined by amplifying the locus with primers surrounding the editing site (black arrows in panel A) that did not overlap in sequence with the repair oligonucleotide. One of the primers had a T3 primer binding site for Sanger sequencing of amplicons (black arrow with flap). Remarkably, after selection, the wild-type allele was not detected ( B ). ( E ) S. rosetta uses repair oligonucleotides with > 20 nt homology arms for genome editing. Truncations of repair oligonucleotides encoding the rpl36a P56Q allele were designed in the same orientation as gRNAs (sense, black dots and lines) or the opposite orientation (antisense, gray dots and lines). 24 hr after S. rosetta recovered from transfections with repair templates and Sp Cas9 RNPs, cycloheximide was added to grow cells in selective media for five days, at which time the cells were harvested for counting cell density and for genotyping. Closed circles indicate that the consensus genotype of the cell population had the rpl36a P56Q allele in Sanger sequencing; whereas, open circles indicate that the cell population had the wild-type allele. E’ and E’’ show two independent trials. Notably, we observed a slight bias for repair oligonucleotides in the sense direction, particularly with shorter homology arms of 20-30 bases. Because repair templates in the sense orientation with 40–80 bases of homologous sequence resulted in the best editing, we performed subsequent optimization with a sense repair oligonucleotide that 50-base homology arms on each side of the double-stranded break. ( F ) Small quantities of Sp Cas9 RNPs are sufficient to initiate genome editing. Decreasing concentrations of Sp Cas9 RNP ( Sp Cas9 was the limiting factor) and a constant amount of repair template were transfected into S. rosetta . After characterizing genome editing outcomes by counting cell density and sequencing the consensus genotype (described in panel E), we found that low concentrations of Sp Cas9 (20 pmol) were sufficient to introduce the rpl36a P56Q allele. F’ and F’’ show two independent trials. ( G ) High concentrations of repair oligonucleotides increase genome editing efficiency. A serial dilution of a repair template was delivered into S. rosetta . The cell density and consensus genotypes from these experiments show that all concentrations of repair template can introduce the rpl36a P56Q allele, but the higher cell densities recovered after transfection with increasing concentrations of repair templates indicate more efficient editing. G’ and G’’ show two independent trials. ( H ) The addition of gRNAs stimulates genome editing. Genome editing was performed by delivering a repair oligonucleotide with Sp Cas9 without the addition of any gRNA or with a gRNA that was prepared from in vitro transcriptions (noted as gRNA in figure) or with a synthetic crRNA that was annealed to a synthetic tracrRNA (noted as crRNA). The consensus genotype and cell densities from these experiments show that gRNAs are necessary for editing and that gRNAs from either source were sufficient for editing. The dots show two independent experiments and lines show their average result.

    Article Snippet: After quantifying DNA (Qubit; Thermo Fisher Scientific) and pooling equimolar amounts of sample, the amplicons were further purified with magnetic beads (UC Berkeley Functional Genomics Lab) and the concentration was verified using qPCR.

    Techniques: Labeling, Sequencing, Mutagenesis, Selection, Binding Assay, Transfection, Introduce, Serial Dilution, In Vitro

    Primer dimer formation in tailed amplicon method. A) Agilent TapeStation trace for a library prepared from samples with N1 and N2 Ct values between ~20-40 using the tailed amplicon v1 (2 pool amplification) workflow. B) Mean read 1 quality score for samples prepared with the tailed amplicon v1 (2 pool amplification) workflow amplified for either 25 or 35 PCR cycles. C) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v1 (2 pool amplification) workflow amplified for either 25 or 35 PCR cycles. D) Agilent Bioanalyzer trace for a library prepared from samples with N1 and N2 Ct values between ~20-35 using the tailed amplicon v2 (4 pool amplification) workflow. E) Mean read 1 quality score for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. F) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. G) 2% agarose gel showing the presence of primer dimers particularly in high N1/N2 Ct samples when indexed using different PCR cycling conditions. Arrow indicates primer dimers on gel.

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Primer dimer formation in tailed amplicon method. A) Agilent TapeStation trace for a library prepared from samples with N1 and N2 Ct values between ~20-40 using the tailed amplicon v1 (2 pool amplification) workflow. B) Mean read 1 quality score for samples prepared with the tailed amplicon v1 (2 pool amplification) workflow amplified for either 25 or 35 PCR cycles. C) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v1 (2 pool amplification) workflow amplified for either 25 or 35 PCR cycles. D) Agilent Bioanalyzer trace for a library prepared from samples with N1 and N2 Ct values between ~20-35 using the tailed amplicon v2 (4 pool amplification) workflow. E) Mean read 1 quality score for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. F) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. G) 2% agarose gel showing the presence of primer dimers particularly in high N1/N2 Ct samples when indexed using different PCR cycling conditions. Arrow indicates primer dimers on gel.

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis

    Tailed amplicon v1 amplicon relative abundance. A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the tailed amplicon v1 protocol at a subsampled read depth of 100,000 raw reads.

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Tailed amplicon v1 amplicon relative abundance. A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the tailed amplicon v1 protocol at a subsampled read depth of 100,000 raw reads.

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Amplification

    Tailed amplicon v2 amplicon relative abundance. A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the tailed amplicon v2 protocol at a subsampled read depth of 100,000 raw reads.

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Tailed amplicon v2 amplicon relative abundance. A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the tailed amplicon v2 protocol at a subsampled read depth of 100,000 raw reads.

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Amplification

    Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on SARS-CoV-2 isolate. A) Percentage of the BEI WA isolate genome coverage at 10x at different subsampled read depths when sequenced with the indicated approach. B) Percent of the BEI WA isolate genome coverage at 100x at different subsampled read depths when sequenced with the indicated approach. C) Observed read depth for each of the expected amplicons for the BEI WA isolate amplified with the ARTIC v3 protocol at a subsampled read depth of 100,000 raw reads. D) Observed read depth for each of the expected amplicons for the BEI WA isolate amplified with the tailed amplicon v1 (2 pool amplification) protocol at a subsampled read depth of 100,000 raw reads. E) Observed read depth for each of the expected amplicons for the BEI WA isolate amplified with the tailed amplicon v2 protocol (4 pool amplification) at a subsampled read depth of 100,000 raw reads. F) Positions of variants detected for the BEI WA isolate at a read depth of up to 1,000,000 raw reads (or the maximum read depth for the sample).

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on SARS-CoV-2 isolate. A) Percentage of the BEI WA isolate genome coverage at 10x at different subsampled read depths when sequenced with the indicated approach. B) Percent of the BEI WA isolate genome coverage at 100x at different subsampled read depths when sequenced with the indicated approach. C) Observed read depth for each of the expected amplicons for the BEI WA isolate amplified with the ARTIC v3 protocol at a subsampled read depth of 100,000 raw reads. D) Observed read depth for each of the expected amplicons for the BEI WA isolate amplified with the tailed amplicon v1 (2 pool amplification) protocol at a subsampled read depth of 100,000 raw reads. E) Observed read depth for each of the expected amplicons for the BEI WA isolate amplified with the tailed amplicon v2 protocol (4 pool amplification) at a subsampled read depth of 100,000 raw reads. F) Positions of variants detected for the BEI WA isolate at a read depth of up to 1,000,000 raw reads (or the maximum read depth for the sample).

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Sequencing, Amplification

    Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on clinical specimens spanning a range of viral loads. A) Samples with N1 and N2 Ct values ranging from approximately 20-35 chosen for testing of SARS-CoV-2 sequencing workflows. B) Evenness of representation of amplicons for different workflows as a function of sample N1 Ct value. Percentage of genome coverage at 100x at different subsampled read depths for each sample when sequenced using the following approaches: D) Illumina Nextera DNA Enrichment; E) ARTIC v3 with TruSeq library preparation. F) Tailed amplicon v1 (2 pool amplification); G) Tailed amplicon v2 (4 pool amplification)

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on clinical specimens spanning a range of viral loads. A) Samples with N1 and N2 Ct values ranging from approximately 20-35 chosen for testing of SARS-CoV-2 sequencing workflows. B) Evenness of representation of amplicons for different workflows as a function of sample N1 Ct value. Percentage of genome coverage at 100x at different subsampled read depths for each sample when sequenced using the following approaches: D) Illumina Nextera DNA Enrichment; E) ARTIC v3 with TruSeq library preparation. F) Tailed amplicon v1 (2 pool amplification); G) Tailed amplicon v2 (4 pool amplification)

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Sequencing, Amplification

    Methods for SARS-CoV-2 genome sequencing compared in this study. A) In Illumina’s Nextera DNA Flex Enrichment protocol cDNA is tagmented and made into barcoded sequencing libraries, which are then enriched using sequence capture with a respiratory virus panel containing probes against SARS-CoV-2. B) In the ARTIC protocol, first strand cDNA is enriched by amplifying with two pools of primers to generate amplicons tiling the SARS-CoV-2 genome. These amplicons are then subjected to either Illumina or Oxford Nanopore library preparation, using methods that either directly add adapters to the ends of the amplicons or fragment them to enable sequencing on a wider variety of Illumina instruments. C) The tailed amplicon approach, developed here, enriches first strand cDNA using ARTIC v3 primers containing adapter tails. This allows functional sequencing libraries to be created through a second indexing PCR reaction that adds sample-specific barcodes and flow cell adapters.

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Methods for SARS-CoV-2 genome sequencing compared in this study. A) In Illumina’s Nextera DNA Flex Enrichment protocol cDNA is tagmented and made into barcoded sequencing libraries, which are then enriched using sequence capture with a respiratory virus panel containing probes against SARS-CoV-2. B) In the ARTIC protocol, first strand cDNA is enriched by amplifying with two pools of primers to generate amplicons tiling the SARS-CoV-2 genome. These amplicons are then subjected to either Illumina or Oxford Nanopore library preparation, using methods that either directly add adapters to the ends of the amplicons or fragment them to enable sequencing on a wider variety of Illumina instruments. C) The tailed amplicon approach, developed here, enriches first strand cDNA using ARTIC v3 primers containing adapter tails. This allows functional sequencing libraries to be created through a second indexing PCR reaction that adds sample-specific barcodes and flow cell adapters.

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Sequencing, Amplification, Functional Assay, Polymerase Chain Reaction

    Coverage metrics by method for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. Percentage of genome coverage at 10x at different subsampled read depths for each sample when sequenced using the following approaches: A) Illumina Nextera DNA Enrichment; B) ARTIC v3 with TruSeq library preparation. C) Tailed amplicon v1 (2 pool amplification); D) Tailed amplicon v2 (4 pool amplification).

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Coverage metrics by method for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. Percentage of genome coverage at 10x at different subsampled read depths for each sample when sequenced using the following approaches: A) Illumina Nextera DNA Enrichment; B) ARTIC v3 with TruSeq library preparation. C) Tailed amplicon v1 (2 pool amplification); D) Tailed amplicon v2 (4 pool amplification).

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Sequencing, Amplification

    Coverage metrics by sample for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. A) Percentage of genome coverage at 10x at different subsampled read depths for the indicated sample when sequenced using the indicated workflow. B) Percentage of genome coverage at 100x at different subsampled read depths for the indicated sample when sequenced using the indicated workflow.

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: Coverage metrics by sample for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. A) Percentage of genome coverage at 10x at different subsampled read depths for the indicated sample when sequenced using the indicated workflow. B) Percentage of genome coverage at 100x at different subsampled read depths for the indicated sample when sequenced using the indicated workflow.

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Sequencing, Amplification

    ARTIC v3 amplicon relative abundance. A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the ARTIC v3 protocol with TruSeq library preparation at a subsampled read depth of 100,000 raw reads.

    Journal: bioRxiv

    Article Title: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

    doi: 10.1101/2020.05.11.088724

    Figure Lengend Snippet: ARTIC v3 amplicon relative abundance. A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the ARTIC v3 protocol with TruSeq library preparation at a subsampled read depth of 100,000 raw reads.

    Article Snippet: Normalization and pooling of tailed amplicon sequencing libraries 10 μL of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Amplification