generacer kit  (Thermo Fisher)


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    Name:
    GeneRacer Kit
    Description:
    GeneRacer is an advanced RACE rapid amplification of cDNA ends technique that improves the efficiency of amplifying full length 5 and 3 cDNA ends With the GeneRacer Kit you can • Generate cDNA from transcripts up to 10 kb in length• Obtain the full length 5 end of rare transcripts at fewer than 30 copies per cell• Clone the full length 5 and 3 ends to construct complete cDNA sequenceThe GeneRacer Kit is available with SuperScript III Reverse Transcriptase RT for improved amplification of the full length 5 end from long and complex mRNA The RNase H portion of SuperScript III RT has been mutated to avoid cleaving mRNA during cDNA synthesis This increases the size and yield of cDNA SuperScript III RT is more thermostable than wild type RTs This enables reverse transcription at higher temperatures relaxing secondary structure of complex templates and allowing cDNA synthesis to go to completion How GeneRacer WorksThe GeneRacer Kit ensures that only transcripts containing full length cDNA ends are amplified 1 2 Figure 1 outlines how the GeneRacer Kit works The advanced protocol starts at the RNA level by specifically targeting only 5 capped mRNA In subsequent steps the cap is removed and replaced with the GeneRacer RNA Oligo During reverse transcription the GeneRacer RNA Oligo sequence is incorporated into the cDNA Only cDNA that is completely reverse transcribed will contain this known sequence 5 RACE PCR is then performed using the GeneRacer 5 Primer specific to the GeneRacer RNA Oligo sequence and a gene specific primer The result is amplified DNA that contains the full length 5 cDNA sequence Sensitivity and LengthTo demonstrate the ability of the GeneRacer Kit to capture the full length 5fi cDNA end the 5fi ends of genes with known transcriptional start sites were amplified Starting with total RNA and following the GeneRacer protocol both long transcripts 10 kb and rare messages present at 0 01 or 30 copies per cell were amplified Figure 2
    Catalog Number:
    l150001
    Price:
    None
    Applications:
    Cloning|PCR & Real-Time PCR|Reverse Transcription|cDNA Libraries & Library Construction
    Category:
    Kits and Assays
    Buy from Supplier


    Structured Review

    Thermo Fisher generacer kit
    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using <t>GeneRacer™</t> 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P
    GeneRacer is an advanced RACE rapid amplification of cDNA ends technique that improves the efficiency of amplifying full length 5 and 3 cDNA ends With the GeneRacer Kit you can • Generate cDNA from transcripts up to 10 kb in length• Obtain the full length 5 end of rare transcripts at fewer than 30 copies per cell• Clone the full length 5 and 3 ends to construct complete cDNA sequenceThe GeneRacer Kit is available with SuperScript III Reverse Transcriptase RT for improved amplification of the full length 5 end from long and complex mRNA The RNase H portion of SuperScript III RT has been mutated to avoid cleaving mRNA during cDNA synthesis This increases the size and yield of cDNA SuperScript III RT is more thermostable than wild type RTs This enables reverse transcription at higher temperatures relaxing secondary structure of complex templates and allowing cDNA synthesis to go to completion How GeneRacer WorksThe GeneRacer Kit ensures that only transcripts containing full length cDNA ends are amplified 1 2 Figure 1 outlines how the GeneRacer Kit works The advanced protocol starts at the RNA level by specifically targeting only 5 capped mRNA In subsequent steps the cap is removed and replaced with the GeneRacer RNA Oligo During reverse transcription the GeneRacer RNA Oligo sequence is incorporated into the cDNA Only cDNA that is completely reverse transcribed will contain this known sequence 5 RACE PCR is then performed using the GeneRacer 5 Primer specific to the GeneRacer RNA Oligo sequence and a gene specific primer The result is amplified DNA that contains the full length 5 cDNA sequence Sensitivity and LengthTo demonstrate the ability of the GeneRacer Kit to capture the full length 5fi cDNA end the 5fi ends of genes with known transcriptional start sites were amplified Starting with total RNA and following the GeneRacer protocol both long transcripts 10 kb and rare messages present at 0 01 or 30 copies per cell were amplified Figure 2
    https://www.bioz.com/result/generacer kit/product/Thermo Fisher
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    Images

    1) Product Images from "Phytophthora infestans Ago1-bound miRNA promotes potato late blight disease"

    Article Title: Phytophthora infestans Ago1-bound miRNA promotes potato late blight disease

    Journal: bioRxiv

    doi: 10.1101/2020.01.28.924175

    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P
    Figure Legend Snippet: StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P

    Techniques Used: Binding Assay, Clone Assay, Infection, Generated, Northern Blot, Labeling, Construct, Luciferase, Reporter Assay, Sequencing

    2) Product Images from "Novel Kinin B1 Receptor Splice Variant and 5?UTR Regulatory Elements Are Responsible for Cell Specific B1 Receptor Expression"

    Article Title: Novel Kinin B1 Receptor Splice Variant and 5?UTR Regulatory Elements Are Responsible for Cell Specific B1 Receptor Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087175

    5′RACE PCR analysis of H2126 cDNA reveals multiple products. H2126 cDNA was amplified using the GeneRacer 5′nested primer and RT Rev 2 primer (A). Expected product size was 450 bp although at least 5 other bands were observed. Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control. Major transcription start sites (TSS) identified in this study are labelled relative to translation start site (ATG) of NCBI published sequences of B 1 R (B). TSS of transcript D identified in this study is located 12 bp upstream of TSS on NCBI (NM_000710) but matches TSS identified by Yang Polgar (1996) [23] . In addition to the full-length wild type B 1 R transcript, a splice variant of B 1 R (transcript D and E) was also identified in this study. The TSS of this splice variant was at two primary locations; 12 bp and 4 bp upstream of NCBI sequence. Schematic presentation of identified wild type (WT) and splice variant (SV) transcripts and position of primers used in RT-qPCR to specifically amplify WT (B1R WT F) and SV (B1R SV F) (C). Forward primers are spanning the splice sites while common reverse primer (B1R Rev qPCR) located in exon 3 was used for amplification of both transcripts.
    Figure Legend Snippet: 5′RACE PCR analysis of H2126 cDNA reveals multiple products. H2126 cDNA was amplified using the GeneRacer 5′nested primer and RT Rev 2 primer (A). Expected product size was 450 bp although at least 5 other bands were observed. Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control. Major transcription start sites (TSS) identified in this study are labelled relative to translation start site (ATG) of NCBI published sequences of B 1 R (B). TSS of transcript D identified in this study is located 12 bp upstream of TSS on NCBI (NM_000710) but matches TSS identified by Yang Polgar (1996) [23] . In addition to the full-length wild type B 1 R transcript, a splice variant of B 1 R (transcript D and E) was also identified in this study. The TSS of this splice variant was at two primary locations; 12 bp and 4 bp upstream of NCBI sequence. Schematic presentation of identified wild type (WT) and splice variant (SV) transcripts and position of primers used in RT-qPCR to specifically amplify WT (B1R WT F) and SV (B1R SV F) (C). Forward primers are spanning the splice sites while common reverse primer (B1R Rev qPCR) located in exon 3 was used for amplification of both transcripts.

    Techniques Used: Polymerase Chain Reaction, Amplification, Variant Assay, Sequencing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    3) Product Images from "Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells"

    Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-50

    The transcription start sites of the reverse transcript from BRCA2 gene bi-directional promoter . (A) GeneRacer amplification product for ZAR2. See Materials and methods for details. (B) Nucleotide sequence of human ZAR2/BRCA2 bi-directional promoter. The transcriptional start sites (TSSs), as determined by GeneRacer technique, are shown. The segment in green color is the sequence complementary to part of the intron 1 sequence of human BRCA2 gene, the segment in red color is from exon 1 and the blue part is from the upstream sequence of BRCA2 gene. The E-box sequence essential for BRCA2 gene expression [ 18 , 19 ] is underlined. The splice donor site at the ZAR2 gene exon 1/intron 1 junction is indicated by a downward arrow. The 'G' residue at the SNP site at -26 from BRCA2 gene transcription start site is shown by a red *. (C) Cartoon showing the human BRCA2 (upper panel) and ZAR2 (lower panel) gene promoter studied. The identities of ZAR2 exon1 (Ex-1) and intron 1 (Int-1) were experimentally determined in this study. TSS: transcription start site (designated as +1); URS: upstream regulatory sequence.
    Figure Legend Snippet: The transcription start sites of the reverse transcript from BRCA2 gene bi-directional promoter . (A) GeneRacer amplification product for ZAR2. See Materials and methods for details. (B) Nucleotide sequence of human ZAR2/BRCA2 bi-directional promoter. The transcriptional start sites (TSSs), as determined by GeneRacer technique, are shown. The segment in green color is the sequence complementary to part of the intron 1 sequence of human BRCA2 gene, the segment in red color is from exon 1 and the blue part is from the upstream sequence of BRCA2 gene. The E-box sequence essential for BRCA2 gene expression [ 18 , 19 ] is underlined. The splice donor site at the ZAR2 gene exon 1/intron 1 junction is indicated by a downward arrow. The 'G' residue at the SNP site at -26 from BRCA2 gene transcription start site is shown by a red *. (C) Cartoon showing the human BRCA2 (upper panel) and ZAR2 (lower panel) gene promoter studied. The identities of ZAR2 exon1 (Ex-1) and intron 1 (Int-1) were experimentally determined in this study. TSS: transcription start site (designated as +1); URS: upstream regulatory sequence.

    Techniques Used: Amplification, Sequencing, Expressing

    4) Product Images from "5?UTR Variants of Ribosomal Protein S19 Transcript Determine Translational Efficiency: Implications for Diamond-Blackfan Anemia and Tissue Variability"

    Article Title: 5?UTR Variants of Ribosomal Protein S19 Transcript Determine Translational Efficiency: Implications for Diamond-Blackfan Anemia and Tissue Variability

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017672

    Transcriptional start sites and tissue expression of RPS19 variants. ( A ) RPS19 5′UTR variants in testis and K562 cells. Schematic presentation of 39 different RPS19 5′UTRs identified of which 29 are yet undescribed. 5′RACE was performed with 1 µg of total RNA using the GeneRacer® kit (Invitrogen) according to manufacturer's recommendation. The RNA was treated with DNase I to clean samples from genomic DNA. The 5′RACE protocol selected full length G-capped mRNA and ruled out the possibility of partially degraded mRNA. PCR products were cloned into a TOPO-TA vector (Invitrogen) and 122 clones were picked randomly (83 from testis, 39 from K562 cells) and analyzed by bidirectional sequencing. The 5′UTR variants identified are indicated and aligned to the first exon of RPS19 from databases with a known maximum 5′UTR of 382 nt (bottom). ( B ) A schematic picture of the 5′ region of RPS19 cDNA (horizontal line) with relative positions of the start codon and the amplicons generated for quantification. Primers used to generate amplicons A, B and C for quantitative PCR are shown as arrows (sequences available upon request). ( C ) Tissue distribution of total RPS19 as determined by qPCR of amplicon A showing relative expression of RPS19 normalized to β-actin on a panel of primary human tissues and cell lines. Analyses were run in triplicates and the average is shown for each tissue. ( D ) Expression of the amplicons B and C representing longer variants of 5′UTR as determined by qPCR and expressed as a percentage of total RPS19 expression determined by amplicon A shown in (C).
    Figure Legend Snippet: Transcriptional start sites and tissue expression of RPS19 variants. ( A ) RPS19 5′UTR variants in testis and K562 cells. Schematic presentation of 39 different RPS19 5′UTRs identified of which 29 are yet undescribed. 5′RACE was performed with 1 µg of total RNA using the GeneRacer® kit (Invitrogen) according to manufacturer's recommendation. The RNA was treated with DNase I to clean samples from genomic DNA. The 5′RACE protocol selected full length G-capped mRNA and ruled out the possibility of partially degraded mRNA. PCR products were cloned into a TOPO-TA vector (Invitrogen) and 122 clones were picked randomly (83 from testis, 39 from K562 cells) and analyzed by bidirectional sequencing. The 5′UTR variants identified are indicated and aligned to the first exon of RPS19 from databases with a known maximum 5′UTR of 382 nt (bottom). ( B ) A schematic picture of the 5′ region of RPS19 cDNA (horizontal line) with relative positions of the start codon and the amplicons generated for quantification. Primers used to generate amplicons A, B and C for quantitative PCR are shown as arrows (sequences available upon request). ( C ) Tissue distribution of total RPS19 as determined by qPCR of amplicon A showing relative expression of RPS19 normalized to β-actin on a panel of primary human tissues and cell lines. Analyses were run in triplicates and the average is shown for each tissue. ( D ) Expression of the amplicons B and C representing longer variants of 5′UTR as determined by qPCR and expressed as a percentage of total RPS19 expression determined by amplicon A shown in (C).

    Techniques Used: Expressing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing, Generated, Real-time Polymerase Chain Reaction, Amplification

    5) Product Images from "Complete Genome Sequence and Analyses of the Subgenomic RNAs of Sweet Potato Chlorotic Stunt Virus Reveal Several New Features for the Genus Crinivirus"

    Article Title: Complete Genome Sequence and Analyses of the Subgenomic RNAs of Sweet Potato Chlorotic Stunt Virus Reveal Several New Features for the Genus Crinivirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.18.9260-9270.2002

    Genomic structure of SPCSV and expression of sgRNAs. (A) The genomic RNA1 and RNA2 are represented by a line, with the ORFs indicated by boxes. ORFs are shown above, below, or in the middle of the line, indicating that they are found in different reading frames. On RNA1, +1 indicates a putative +1 ribosomal frameshift site. The functional domains predicted from the deduced amino acid sequence are indicated inside the boxes, or, if no function could be predicted, the approximate molecular weight is indicated. P-Pro, putative papain-like leader proteinase, for which the arrow and dotted line indicate the predicted autocatalytic cleavage site; MTR, methyltransferase domain; HEL, helicase domain; RdRp, RNA-dependant RNA polymerase domain; RNase3, RNase III-like domain; Hsp70h, heat shock protein 70 family homologue; CP, coat protein; CPm, minor coat protein. The lines below the genomic RNA indicate sgRNAs, each of which was named according to the first ORF at the 5′ end. Dashed lines indicate hypothetical sgRNAs that were not detected with the GeneRacer method. Arrowheads indicate the positions of primers used for amplification of the 5′ ends from the genomic and sgRNAs with GeneRacer, the products of which are shown in panel B (2% agarose gel; positions of molecular size markers [in nucleotides] are shown to the left). Grey arrowheads in panel A indicate that no product was obtained in GeneRacer with these primers. (C) The 5′ untranslated sequences of the sgRNAs are shown, as determined from PCR products obtained with GeneRacer. Amplification of the p60 sgRNA 5′ end resulted in two fragments, of which one was 53 nt shorter but otherwise identical.
    Figure Legend Snippet: Genomic structure of SPCSV and expression of sgRNAs. (A) The genomic RNA1 and RNA2 are represented by a line, with the ORFs indicated by boxes. ORFs are shown above, below, or in the middle of the line, indicating that they are found in different reading frames. On RNA1, +1 indicates a putative +1 ribosomal frameshift site. The functional domains predicted from the deduced amino acid sequence are indicated inside the boxes, or, if no function could be predicted, the approximate molecular weight is indicated. P-Pro, putative papain-like leader proteinase, for which the arrow and dotted line indicate the predicted autocatalytic cleavage site; MTR, methyltransferase domain; HEL, helicase domain; RdRp, RNA-dependant RNA polymerase domain; RNase3, RNase III-like domain; Hsp70h, heat shock protein 70 family homologue; CP, coat protein; CPm, minor coat protein. The lines below the genomic RNA indicate sgRNAs, each of which was named according to the first ORF at the 5′ end. Dashed lines indicate hypothetical sgRNAs that were not detected with the GeneRacer method. Arrowheads indicate the positions of primers used for amplification of the 5′ ends from the genomic and sgRNAs with GeneRacer, the products of which are shown in panel B (2% agarose gel; positions of molecular size markers [in nucleotides] are shown to the left). Grey arrowheads in panel A indicate that no product was obtained in GeneRacer with these primers. (C) The 5′ untranslated sequences of the sgRNAs are shown, as determined from PCR products obtained with GeneRacer. Amplification of the p60 sgRNA 5′ end resulted in two fragments, of which one was 53 nt shorter but otherwise identical.

    Techniques Used: Expressing, Functional Assay, Sequencing, Molecular Weight, Amplification, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    6) Product Images from "Allele-specific siRNA silencing for the common Keratin 12 founder mutation in Meesmann epithelial corneal dystrophy"

    Article Title: Allele-specific siRNA silencing for the common Keratin 12 founder mutation in Meesmann epithelial corneal dystrophy

    Journal: Investigative ophthalmology & visual science

    doi: 10.1167/iovs.12-10528

    5′RACE verifies siRNA-mediated cleavage at the predicted site on K12 mRNA K12 specific mRNA cleavage products of AD293 cells exogenously expressing K12-Arg135Thr and treated with 3nM siRNA K12-Arg135Thr-5 were assessed by a modified 5′ Rapid Amplification of cDNA Ends (5′RACE). The generacer oligo adapter was ligated to the free 5′ end of cleaved mRNA products. K12 specific cleaved mRNAs were selected by PCR amplification with a K12 Gene Specific Primer (GSP). The major K12 specific PCR product was purified, ligated into pCR4.0 topo plasmid and sequenced. Sequencing shows that the major product had the generacer adapter ligated to the 399 th nucleotide of the K12 CDS, this corresponding to the predicted cleavage site between nucleotides 10 and 11 of the hybridized guide strand of siRNA K12-Arg135Thr-5.
    Figure Legend Snippet: 5′RACE verifies siRNA-mediated cleavage at the predicted site on K12 mRNA K12 specific mRNA cleavage products of AD293 cells exogenously expressing K12-Arg135Thr and treated with 3nM siRNA K12-Arg135Thr-5 were assessed by a modified 5′ Rapid Amplification of cDNA Ends (5′RACE). The generacer oligo adapter was ligated to the free 5′ end of cleaved mRNA products. K12 specific cleaved mRNAs were selected by PCR amplification with a K12 Gene Specific Primer (GSP). The major K12 specific PCR product was purified, ligated into pCR4.0 topo plasmid and sequenced. Sequencing shows that the major product had the generacer adapter ligated to the 399 th nucleotide of the K12 CDS, this corresponding to the predicted cleavage site between nucleotides 10 and 11 of the hybridized guide strand of siRNA K12-Arg135Thr-5.

    Techniques Used: Expressing, Modification, Rapid Amplification of cDNA Ends, Polymerase Chain Reaction, Amplification, Purification, Plasmid Preparation, Sequencing

    7) Product Images from "Characterization of the angiotensin (AT1b) receptor promoter and its regulation by glucocorticoids"

    Article Title: Characterization of the angiotensin (AT1b) receptor promoter and its regulation by glucocorticoids

    Journal: Journal of Molecular Endocrinology

    doi: 10.1677/JME-09-0036

    Mapping of the Agtr1b promoter. (a) Agarose gel showing the products of the 5′ RACE. Two major products (a and b) are found using independent reverse gene specific primers located at positions 639–654 (GSP1) and 537–559 (GSP2) of the Genebank genomic sequence S69961 . Sequence analysis showed that the majority of clones derived from the ‘a’ band mapped a start site equivalent to position 1437 of the Genebank 5′ region and exon 1 sequence U01033 . GR5′ is GeneRacer 5′ primer and GRn is GeneRacer nested primer from Invitrogen. (b) A diagrammatic representation of 1·3 kbp of promoter indicating the location of more distal GREs. Three CpGs sites located within the Sp1-2 and Sp1-3 are shown as closed circles. (c) DNA sequence of the putative proximal promoter showing the transcriptional start site (+1), the probable TATA, CAAT box (both underlined), GRE4, AP1-1 and -2 (gray boxes), and three SP1 sites. Vertical arrows labeled del 202 and del 85 represent the 5′ ends of the two deletion constructs of the promoter studied in luciferase assays.
    Figure Legend Snippet: Mapping of the Agtr1b promoter. (a) Agarose gel showing the products of the 5′ RACE. Two major products (a and b) are found using independent reverse gene specific primers located at positions 639–654 (GSP1) and 537–559 (GSP2) of the Genebank genomic sequence S69961 . Sequence analysis showed that the majority of clones derived from the ‘a’ band mapped a start site equivalent to position 1437 of the Genebank 5′ region and exon 1 sequence U01033 . GR5′ is GeneRacer 5′ primer and GRn is GeneRacer nested primer from Invitrogen. (b) A diagrammatic representation of 1·3 kbp of promoter indicating the location of more distal GREs. Three CpGs sites located within the Sp1-2 and Sp1-3 are shown as closed circles. (c) DNA sequence of the putative proximal promoter showing the transcriptional start site (+1), the probable TATA, CAAT box (both underlined), GRE4, AP1-1 and -2 (gray boxes), and three SP1 sites. Vertical arrows labeled del 202 and del 85 represent the 5′ ends of the two deletion constructs of the promoter studied in luciferase assays.

    Techniques Used: Agarose Gel Electrophoresis, Sequencing, Clone Assay, Derivative Assay, Labeling, Construct, Luciferase

    8) Product Images from "H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts"

    Article Title: H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037923

    Characterisation of TSS of the endogenous mouse 91H RNA. 5′RACE experiment was performed on unpolyadenylated and capped RNA from 7 days-old mouse liver. (A) The RT Primer was designed in the mFb region ( Figure 1 ) and a band was successfully amplified by nested PCRs. The RT primer corresponds with the forward primer of PCRa and nested PCR reactions were performed using the GeneRacer DNA oligonucleotide as reverse primer. (B) Ethidium bromide staining of an agarose gel showing PCRs product obtained from amplifications indicated above (MW: Molecular Weight). Sequencing of PCRa and PCRc products showed that these bands correspond essentially to unspecific amplifications while PCRb correspond to the TSS of the 91H RNA. (C) Electrophoregram of the sequenced 5′RACE product amplified from the capped RNA fraction (PCRb). This sequence identified a unique Cap site located in the endodermic enhancer 2 at position chr7:149,755,206 or chr7:149,755,207 on mouse July 2007/ mm9 Assembly. Due to the presence of a C residue at the end of the GeneRacer RNA oligonucleotide primer and/or the possibility that the last C residue may derive from the cap of the RNA, the exact position of the TSS remains ambiguous between two consecutive C residues found in the mouse genome sequence. (D) The sequence of the endodermic enhancer 2 is indicated in bold. The position of the TSS of the 91H RNA is indicated (black arrow).
    Figure Legend Snippet: Characterisation of TSS of the endogenous mouse 91H RNA. 5′RACE experiment was performed on unpolyadenylated and capped RNA from 7 days-old mouse liver. (A) The RT Primer was designed in the mFb region ( Figure 1 ) and a band was successfully amplified by nested PCRs. The RT primer corresponds with the forward primer of PCRa and nested PCR reactions were performed using the GeneRacer DNA oligonucleotide as reverse primer. (B) Ethidium bromide staining of an agarose gel showing PCRs product obtained from amplifications indicated above (MW: Molecular Weight). Sequencing of PCRa and PCRc products showed that these bands correspond essentially to unspecific amplifications while PCRb correspond to the TSS of the 91H RNA. (C) Electrophoregram of the sequenced 5′RACE product amplified from the capped RNA fraction (PCRb). This sequence identified a unique Cap site located in the endodermic enhancer 2 at position chr7:149,755,206 or chr7:149,755,207 on mouse July 2007/ mm9 Assembly. Due to the presence of a C residue at the end of the GeneRacer RNA oligonucleotide primer and/or the possibility that the last C residue may derive from the cap of the RNA, the exact position of the TSS remains ambiguous between two consecutive C residues found in the mouse genome sequence. (D) The sequence of the endodermic enhancer 2 is indicated in bold. The position of the TSS of the 91H RNA is indicated (black arrow).

    Techniques Used: Amplification, Nested PCR, Staining, Agarose Gel Electrophoresis, Molecular Weight, Sequencing

    9) Product Images from "Tobacco rattle virus-induced PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH) gene silencing in Solanum pseudocapsicum L."

    Article Title: Tobacco rattle virus-induced PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH) gene silencing in Solanum pseudocapsicum L.

    Journal: PeerJ

    doi: 10.7717/peerj.4424

    Cloning of the reference genes and PDS and ChlH gene fragments in S. pseudocapsicum . Amplification of SpACTIN , SpGAPDH , SpUBQ , SpPDS and SPChlH from S. pseudocapsicum leaves. The primers for SpACTIN , SpGAPDH and SpUBQ were designed from conserved regions of ACTIN , GAPDH and UBQ based on the alignment of tomato, pepper, tobacco and potato CDS sequences. The primers for SpPDS and SPChlH were designed from conserved regions of PDS and ChlH based on the alignment of tomato, pepper, and tobacco CDS sequences. The cDNA from S. pseudocapsicum leaves was used as the template for the amplification of the corresponding PCR products. Lane 1, RT-PCR products of the ACTIN gene; Lane 2, RT-PCR product of the GAPDH gene; Lane 3, RT-PCR product of the UBQ gene; Lane 4, RT-PCR product of the ChlH gene; Lane 5, RT-PCR product of the PDS gene; and Lane 6, 3′RACE product of the PDS gene. The gene-specific primers (GSPs) for 3′RACE were designed based on the sequence of Lane 5 according to the manufacturer’s instructions provided with the GeneRacer™ kit (Invitrogen, Carlsbad, CA, USA). All the primers are shown in Table S1 .
    Figure Legend Snippet: Cloning of the reference genes and PDS and ChlH gene fragments in S. pseudocapsicum . Amplification of SpACTIN , SpGAPDH , SpUBQ , SpPDS and SPChlH from S. pseudocapsicum leaves. The primers for SpACTIN , SpGAPDH and SpUBQ were designed from conserved regions of ACTIN , GAPDH and UBQ based on the alignment of tomato, pepper, tobacco and potato CDS sequences. The primers for SpPDS and SPChlH were designed from conserved regions of PDS and ChlH based on the alignment of tomato, pepper, and tobacco CDS sequences. The cDNA from S. pseudocapsicum leaves was used as the template for the amplification of the corresponding PCR products. Lane 1, RT-PCR products of the ACTIN gene; Lane 2, RT-PCR product of the GAPDH gene; Lane 3, RT-PCR product of the UBQ gene; Lane 4, RT-PCR product of the ChlH gene; Lane 5, RT-PCR product of the PDS gene; and Lane 6, 3′RACE product of the PDS gene. The gene-specific primers (GSPs) for 3′RACE were designed based on the sequence of Lane 5 according to the manufacturer’s instructions provided with the GeneRacer™ kit (Invitrogen, Carlsbad, CA, USA). All the primers are shown in Table S1 .

    Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    10) Product Images from "Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1"

    Article Title: Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg914

    ( A ) Single-stranded DNA ligation using the phosphatase-resistant assay, using 10 µM 32 P-d(A 20 ) as template, showing the accumulation of products over extended periods of time. ( B ) Circularization of P-d(A 20 ) using 0.1 and 0.2 mg/ml enzyme concentration with 1 µM (white bar) and 2.5 µM (black bar) template with and without 1 mM HCC and 25% PEG6000. The additives increase the activity 3–4-fold, reaching the maximum of 20% after 8 h at 60°C. ( C ) A Lineweaver–Burk plot, derived from adenylation of 32 P-r(A 10 ) 3′-dideoxy-blocked oligomer, was measured using ATP titration. ATP inhibited adenylation at > 100 µM ATP concentration. ( D ) RACE experiment using vector-derived RNA template and components from the Generacer™ kit (Invitrogen Inc.) followed by cDNA synthesis and PCR. RM378 RNA ligase 1 and buffer were used instead of T4 RNA ligase 1, and were incubated at 60°C for 1 h. Results show that 300-bp PCR products were obtained from the sample at different dilutions.
    Figure Legend Snippet: ( A ) Single-stranded DNA ligation using the phosphatase-resistant assay, using 10 µM 32 P-d(A 20 ) as template, showing the accumulation of products over extended periods of time. ( B ) Circularization of P-d(A 20 ) using 0.1 and 0.2 mg/ml enzyme concentration with 1 µM (white bar) and 2.5 µM (black bar) template with and without 1 mM HCC and 25% PEG6000. The additives increase the activity 3–4-fold, reaching the maximum of 20% after 8 h at 60°C. ( C ) A Lineweaver–Burk plot, derived from adenylation of 32 P-r(A 10 ) 3′-dideoxy-blocked oligomer, was measured using ATP titration. ATP inhibited adenylation at > 100 µM ATP concentration. ( D ) RACE experiment using vector-derived RNA template and components from the Generacer™ kit (Invitrogen Inc.) followed by cDNA synthesis and PCR. RM378 RNA ligase 1 and buffer were used instead of T4 RNA ligase 1, and were incubated at 60°C for 1 h. Results show that 300-bp PCR products were obtained from the sample at different dilutions.

    Techniques Used: DNA Ligation, Concentration Assay, Activity Assay, Derivative Assay, Titration, Plasmid Preparation, Polymerase Chain Reaction, Incubation

    11) Product Images from "Novel Kinin B1 Receptor Splice Variant and 5?UTR Regulatory Elements Are Responsible for Cell Specific B1 Receptor Expression"

    Article Title: Novel Kinin B1 Receptor Splice Variant and 5?UTR Regulatory Elements Are Responsible for Cell Specific B1 Receptor Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087175

    5′RACE PCR analysis of H2126 cDNA reveals multiple products. H2126 cDNA was amplified using the GeneRacer 5′nested primer and RT Rev 2 primer (A). Expected product size was 450 bp although at least 5 other bands were observed. Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control. Major transcription start sites (TSS) identified in this study are labelled relative to translation start site (ATG) of NCBI published sequences of B 1 R (B). TSS of transcript D identified in this study is located 12 bp upstream of TSS on NCBI (NM_000710) but matches TSS identified by Yang Polgar (1996) [23] . In addition to the full-length wild type B 1 R transcript, a splice variant of B 1 R (transcript D and E) was also identified in this study. The TSS of this splice variant was at two primary locations; 12 bp and 4 bp upstream of NCBI sequence. Schematic presentation of identified wild type (WT) and splice variant (SV) transcripts and position of primers used in RT-qPCR to specifically amplify WT (B1R WT F) and SV (B1R SV F) (C). Forward primers are spanning the splice sites while common reverse primer (B1R Rev qPCR) located in exon 3 was used for amplification of both transcripts.
    Figure Legend Snippet: 5′RACE PCR analysis of H2126 cDNA reveals multiple products. H2126 cDNA was amplified using the GeneRacer 5′nested primer and RT Rev 2 primer (A). Expected product size was 450 bp although at least 5 other bands were observed. Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control. Major transcription start sites (TSS) identified in this study are labelled relative to translation start site (ATG) of NCBI published sequences of B 1 R (B). TSS of transcript D identified in this study is located 12 bp upstream of TSS on NCBI (NM_000710) but matches TSS identified by Yang Polgar (1996) [23] . In addition to the full-length wild type B 1 R transcript, a splice variant of B 1 R (transcript D and E) was also identified in this study. The TSS of this splice variant was at two primary locations; 12 bp and 4 bp upstream of NCBI sequence. Schematic presentation of identified wild type (WT) and splice variant (SV) transcripts and position of primers used in RT-qPCR to specifically amplify WT (B1R WT F) and SV (B1R SV F) (C). Forward primers are spanning the splice sites while common reverse primer (B1R Rev qPCR) located in exon 3 was used for amplification of both transcripts.

    Techniques Used: Polymerase Chain Reaction, Amplification, Variant Assay, Sequencing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    12) Product Images from "Unexpected Transcripts in Tn7 orf19.2646 C. albicans Mutant Lead to Low Fungal Burden Phenotype In vivo"

    Article Title: Unexpected Transcripts in Tn7 orf19.2646 C. albicans Mutant Lead to Low Fungal Burden Phenotype In vivo

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00873

    RACE analysis. (A) Map of Tn7 mutated orf19.2646 . Orf19.2646 (gray) is interrupted by the Tn7-UAU cassette (white bar). The putative transcripts drawn between the dotted lines have been ligated to a 5′ GeneRacer™ Oligo and 3′ GeneRacer™ Oligo dT primers (purple). The forward primers (P1, P1b, and P2b, red triangles) are designed to amplify a products with the GeneRacer 3′ primer (GR 3′), while the reverse primers (P3, P3b, P4, and P4b, blue triangles) are designed to amplify products with the GeneRacer 5′ primer (GR 5′). (B) Left panel: Control PCR with HeLa total RNA. Middle panel: RACE PCRs performed with the GR5′, the GR3′ primers and with the P1-P4 primers on SC5314 RNA as control of reverse transcription. Right panel: Nested PCR performed on the GR5-P4 and GR5-P3 PCR products (black arrows). Topo cloning was performed on the products indicated by a white arrow.
    Figure Legend Snippet: RACE analysis. (A) Map of Tn7 mutated orf19.2646 . Orf19.2646 (gray) is interrupted by the Tn7-UAU cassette (white bar). The putative transcripts drawn between the dotted lines have been ligated to a 5′ GeneRacer™ Oligo and 3′ GeneRacer™ Oligo dT primers (purple). The forward primers (P1, P1b, and P2b, red triangles) are designed to amplify a products with the GeneRacer 3′ primer (GR 3′), while the reverse primers (P3, P3b, P4, and P4b, blue triangles) are designed to amplify products with the GeneRacer 5′ primer (GR 5′). (B) Left panel: Control PCR with HeLa total RNA. Middle panel: RACE PCRs performed with the GR5′, the GR3′ primers and with the P1-P4 primers on SC5314 RNA as control of reverse transcription. Right panel: Nested PCR performed on the GR5-P4 and GR5-P3 PCR products (black arrows). Topo cloning was performed on the products indicated by a white arrow.

    Techniques Used: Polymerase Chain Reaction, Nested PCR, Clone Assay

    13) Product Images from "Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8) *"

    Article Title: Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.123158

    TSS of Mct8 in F9 cells. A , results of 5′-RACE from tRA-treated F9 cells. Shown is agarose gel electrophoresis of the second round PCR with GeneRacer 5′ nested primer and 3′ +496 primer. Marker ( M ) was a 100-bp ladder. B , distribution
    Figure Legend Snippet: TSS of Mct8 in F9 cells. A , results of 5′-RACE from tRA-treated F9 cells. Shown is agarose gel electrophoresis of the second round PCR with GeneRacer 5′ nested primer and 3′ +496 primer. Marker ( M ) was a 100-bp ladder. B , distribution

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    14) Product Images from "The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning"

    Article Title: The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160510242

    Analysis of the putative Snama promoter. ( A ) Schematic representations of the Snama genomic DNA starting at the putative transcriptional start site (TSS), the cDNA expected from rapid amplification of cDNA ends (RACE) and the domain with no name (DWNN) catalytic module. Arrows indicate primers used as specified in the Materials and Methods; E = exon, I = intron ( B ) 5' RACE products using Drosophila mRNA: (i) Amplification using combinations of GeneRacer 5' nested and GeneRacer 5' forward primers together with the RING finger (RF) tail and RF tail internal as reverse primers gives rise to a ~800-bp product (Lanes 2 and 4), and a smaller product of ~650 bp (indicated by an asterisk) was amplified when the GeneRacer 5' nested and GeneRacer 5' forward primers are combined with RF tail reverse primer (Lanes 1 and 3). A positive control showing the amplification of the RACE-ready β-actin cDNA, created from HeLa RNA, results in a ~900-bp fragment, as expected (Lanes 5 and 6). The difference in size between the β-actin cDNA amplification product with GR 5' nested primer (858 bp) and GR 5' primer (872 bp) is only 14 bp; (ii) Cloning and PCR amplification of the 5' RACE products: Lane 1 shows the smaller ~650-bp cloned fragment (asterisk), whilst Lanes 2 and 3 show the larger ~800-bp cloned fragment; ( C ) Sequence of the putative promoter region. The underlined nucleotide is the putative transcriptional start site; ( D ) (i) Schematic representation of fragments (P1–P3) of the promoter region showing base positions relative to the transcription start site (+1); (ii) Dual luciferase assay to determine the maximal promoter sequence. Measurement of luminescence activity of firefly and Renilla luciferase activity indicated that the maximal promoter sequence required to drive Snama transcription was Promoter 2; ( E ) Mobility shift assay of biotin-labelled and unlabeled DNA Snama promoter DNA. Lanes 1–3 indicate the Epstein–Barr nuclear antigen (EBNA) control system, biotin-EBNA control DNA, biotin-EBNA control DNA and EBNA extract and biotin-EBNA control DNA and EBNA extracted with excess unlabeled EBNA DNA, respectively. The biotin-EBNA control DNA shows no shift; the biotin-EBNA control DNA and EBNA extract shows a shift; and the biotin-EBNA control DNA and EBNA extract and excess unlabeled EBNA DNA show minimal shift due to competition. Lanes 4–6 show the unlabeled DNA fragments P1, P2 and P3. Lanes 7–9 represent labelled promoters P1, P2 and P3 and a mobility shift due to the binding of nuclear proteins to the promoters; ( F ) Electromobility shift assay (EMSA) of biotin-labelled protein-DNA complexes separated from crude extracts by streptavidin affinity chromatography. Promoter 0 represents the −231 region. (i) Increasing concentrations of labelled Promoter 2 DNA were used to elute binding proteins from embryonic nuclear extracts; (ii) Four micrograms of promoter DNA as indicated were used in order to select the best promoter to use in (i). Arrows indicate the bound nuclear proteins that were selected for identification by mass spectrometry; ( G ) Schematic representation of the (−673; +49) sequence shows the predicted transcription factor binding sites.
    Figure Legend Snippet: Analysis of the putative Snama promoter. ( A ) Schematic representations of the Snama genomic DNA starting at the putative transcriptional start site (TSS), the cDNA expected from rapid amplification of cDNA ends (RACE) and the domain with no name (DWNN) catalytic module. Arrows indicate primers used as specified in the Materials and Methods; E = exon, I = intron ( B ) 5' RACE products using Drosophila mRNA: (i) Amplification using combinations of GeneRacer 5' nested and GeneRacer 5' forward primers together with the RING finger (RF) tail and RF tail internal as reverse primers gives rise to a ~800-bp product (Lanes 2 and 4), and a smaller product of ~650 bp (indicated by an asterisk) was amplified when the GeneRacer 5' nested and GeneRacer 5' forward primers are combined with RF tail reverse primer (Lanes 1 and 3). A positive control showing the amplification of the RACE-ready β-actin cDNA, created from HeLa RNA, results in a ~900-bp fragment, as expected (Lanes 5 and 6). The difference in size between the β-actin cDNA amplification product with GR 5' nested primer (858 bp) and GR 5' primer (872 bp) is only 14 bp; (ii) Cloning and PCR amplification of the 5' RACE products: Lane 1 shows the smaller ~650-bp cloned fragment (asterisk), whilst Lanes 2 and 3 show the larger ~800-bp cloned fragment; ( C ) Sequence of the putative promoter region. The underlined nucleotide is the putative transcriptional start site; ( D ) (i) Schematic representation of fragments (P1–P3) of the promoter region showing base positions relative to the transcription start site (+1); (ii) Dual luciferase assay to determine the maximal promoter sequence. Measurement of luminescence activity of firefly and Renilla luciferase activity indicated that the maximal promoter sequence required to drive Snama transcription was Promoter 2; ( E ) Mobility shift assay of biotin-labelled and unlabeled DNA Snama promoter DNA. Lanes 1–3 indicate the Epstein–Barr nuclear antigen (EBNA) control system, biotin-EBNA control DNA, biotin-EBNA control DNA and EBNA extract and biotin-EBNA control DNA and EBNA extracted with excess unlabeled EBNA DNA, respectively. The biotin-EBNA control DNA shows no shift; the biotin-EBNA control DNA and EBNA extract shows a shift; and the biotin-EBNA control DNA and EBNA extract and excess unlabeled EBNA DNA show minimal shift due to competition. Lanes 4–6 show the unlabeled DNA fragments P1, P2 and P3. Lanes 7–9 represent labelled promoters P1, P2 and P3 and a mobility shift due to the binding of nuclear proteins to the promoters; ( F ) Electromobility shift assay (EMSA) of biotin-labelled protein-DNA complexes separated from crude extracts by streptavidin affinity chromatography. Promoter 0 represents the −231 region. (i) Increasing concentrations of labelled Promoter 2 DNA were used to elute binding proteins from embryonic nuclear extracts; (ii) Four micrograms of promoter DNA as indicated were used in order to select the best promoter to use in (i). Arrows indicate the bound nuclear proteins that were selected for identification by mass spectrometry; ( G ) Schematic representation of the (−673; +49) sequence shows the predicted transcription factor binding sites.

    Techniques Used: Rapid Amplification of cDNA Ends, Amplification, Positive Control, Clone Assay, Polymerase Chain Reaction, Sequencing, Luciferase, Activity Assay, Mobility Shift, Binding Assay, Electro Mobility Shift Assay, Affinity Chromatography, Mass Spectrometry

    15) Product Images from "Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1"

    Article Title: Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg914

    ( A ) Single-stranded DNA ligation using the phosphatase-resistant assay, using 10 µM 32 P-d(A 20 ) as template, showing the accumulation of products over extended periods of time. ( B ) Circularization of P-d(A 20 ) using 0.1 and 0.2 mg/ml enzyme concentration with 1 µM (white bar) and 2.5 µM (black bar) template with and without 1 mM HCC and 25% PEG6000. The additives increase the activity 3–4-fold, reaching the maximum of 20% after 8 h at 60°C. ( C ) A Lineweaver–Burk plot, derived from adenylation of 32 P-r(A 10 ) 3′-dideoxy-blocked oligomer, was measured using ATP titration. ATP inhibited adenylation at > 100 µM ATP concentration. ( D ) RACE experiment using vector-derived RNA template and components from the Generacer™ kit (Invitrogen Inc.) followed by cDNA synthesis and PCR. RM378 RNA ligase 1 and buffer were used instead of T4 RNA ligase 1, and were incubated at 60°C for 1 h. Results show that 300-bp PCR products were obtained from the sample at different dilutions.
    Figure Legend Snippet: ( A ) Single-stranded DNA ligation using the phosphatase-resistant assay, using 10 µM 32 P-d(A 20 ) as template, showing the accumulation of products over extended periods of time. ( B ) Circularization of P-d(A 20 ) using 0.1 and 0.2 mg/ml enzyme concentration with 1 µM (white bar) and 2.5 µM (black bar) template with and without 1 mM HCC and 25% PEG6000. The additives increase the activity 3–4-fold, reaching the maximum of 20% after 8 h at 60°C. ( C ) A Lineweaver–Burk plot, derived from adenylation of 32 P-r(A 10 ) 3′-dideoxy-blocked oligomer, was measured using ATP titration. ATP inhibited adenylation at > 100 µM ATP concentration. ( D ) RACE experiment using vector-derived RNA template and components from the Generacer™ kit (Invitrogen Inc.) followed by cDNA synthesis and PCR. RM378 RNA ligase 1 and buffer were used instead of T4 RNA ligase 1, and were incubated at 60°C for 1 h. Results show that 300-bp PCR products were obtained from the sample at different dilutions.

    Techniques Used: DNA Ligation, Concentration Assay, Activity Assay, Derivative Assay, Titration, Plasmid Preparation, Polymerase Chain Reaction, Incubation

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    Isolation:

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    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using <t>GeneRacer™</t> 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P
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    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P

    Journal: bioRxiv

    Article Title: Phytophthora infestans Ago1-bound miRNA promotes potato late blight disease

    doi: 10.1101/2020.01.28.924175

    Figure Lengend Snippet: StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P

    Article Snippet: First, a touch down PCR with GeneRacer™ 5′ primer and a gene-specific reverse primer was run.

    Techniques: Binding Assay, Clone Assay, Infection, Generated, Northern Blot, Labeling, Construct, Luciferase, Reporter Assay, Sequencing

    RACE analysis. (A) Map of Tn7 mutated orf19.2646 . Orf19.2646 (gray) is interrupted by the Tn7-UAU cassette (white bar). The putative transcripts drawn between the dotted lines have been ligated to a 5′ GeneRacer™ Oligo and 3′ GeneRacer™ Oligo dT primers (purple). The forward primers (P1, P1b, and P2b, red triangles) are designed to amplify a products with the GeneRacer 3′ primer (GR 3′), while the reverse primers (P3, P3b, P4, and P4b, blue triangles) are designed to amplify products with the GeneRacer 5′ primer (GR 5′). (B) Left panel: Control PCR with HeLa total RNA. Middle panel: RACE PCRs performed with the GR5′, the GR3′ primers and with the P1-P4 primers on SC5314 RNA as control of reverse transcription. Right panel: Nested PCR performed on the GR5-P4 and GR5-P3 PCR products (black arrows). Topo cloning was performed on the products indicated by a white arrow.

    Journal: Frontiers in Microbiology

    Article Title: Unexpected Transcripts in Tn7 orf19.2646 C. albicans Mutant Lead to Low Fungal Burden Phenotype In vivo

    doi: 10.3389/fmicb.2017.00873

    Figure Lengend Snippet: RACE analysis. (A) Map of Tn7 mutated orf19.2646 . Orf19.2646 (gray) is interrupted by the Tn7-UAU cassette (white bar). The putative transcripts drawn between the dotted lines have been ligated to a 5′ GeneRacer™ Oligo and 3′ GeneRacer™ Oligo dT primers (purple). The forward primers (P1, P1b, and P2b, red triangles) are designed to amplify a products with the GeneRacer 3′ primer (GR 3′), while the reverse primers (P3, P3b, P4, and P4b, blue triangles) are designed to amplify products with the GeneRacer 5′ primer (GR 5′). (B) Left panel: Control PCR with HeLa total RNA. Middle panel: RACE PCRs performed with the GR5′, the GR3′ primers and with the P1-P4 primers on SC5314 RNA as control of reverse transcription. Right panel: Nested PCR performed on the GR5-P4 and GR5-P3 PCR products (black arrows). Topo cloning was performed on the products indicated by a white arrow.

    Article Snippet: RNA ligase-mediated-rapid amplification of cDNA ends (RLM-RACE) The RLM-RACE was performed according to manufacturer's protocol (GeneRacer™ Kit, Thermofisher, Switzerland).

    Techniques: Polymerase Chain Reaction, Nested PCR, Clone Assay