Structured Review

MACHEREY NAGEL pcr clean up
Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
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1) Product Images from "In vivo targeted single-nucleotide editing in zebrafish"

Article Title: In vivo targeted single-nucleotide editing in zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-018-29794-9

Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
Figure Legend Snippet: Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

Techniques Used: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing

2) Product Images from "A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus Escape Mutations in Non-human Primates"

Article Title: A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus Escape Mutations in Non-human Primates

Journal: Cell reports

doi: 10.1016/j.celrep.2018.10.031

Administration of Z004 Alone Leads to the Emergence of Resistant ZIKV in Rhesus Macaques ( Macaca mulatta ) (A) Macaques were administered monoclonal antibody Z004 (red) 1 day before intravenous inoculation with 10 5 PFU of ZIKV or were untreated (black). The graph shows plasma viral RNA levels of ZIKV over time as determined by qRT-PCR. (B) ZIKV mutations emerging in macaques treated with Z004 alone. An alignment in the region of residues E393-K394 (bold) within the EDIII is shown at the top, and chromatograms of the PCR amplicons showing the mutations (indicated by red arrows) are shown at the bottom. Amino acid changes compared to the inoculum sequence are highlighted in red. In macaque 6414, E393D and K394R are on separate viral RNAs, as determined by sequencing of the cloned amplicon. (C) The epitope of ZIKV EDIII recognized by the Z004-related antibody Z006 is shown in red (PDB: 5VIG). The E393-K394 residues are highlighted. (D) Impaired binding of Z004 to the EDIII escape mutants. ELISA demonstrates reduced binding of Z004 Fab to EDIII K394R and EDIII E393D . The positive control antibody Z015 recognizes an epitope that is independent of the E393 and K394 residues. Data are presented as mean ± SD of triplicates, and a representative of two experiments is shown. .
Figure Legend Snippet: Administration of Z004 Alone Leads to the Emergence of Resistant ZIKV in Rhesus Macaques ( Macaca mulatta ) (A) Macaques were administered monoclonal antibody Z004 (red) 1 day before intravenous inoculation with 10 5 PFU of ZIKV or were untreated (black). The graph shows plasma viral RNA levels of ZIKV over time as determined by qRT-PCR. (B) ZIKV mutations emerging in macaques treated with Z004 alone. An alignment in the region of residues E393-K394 (bold) within the EDIII is shown at the top, and chromatograms of the PCR amplicons showing the mutations (indicated by red arrows) are shown at the bottom. Amino acid changes compared to the inoculum sequence are highlighted in red. In macaque 6414, E393D and K394R are on separate viral RNAs, as determined by sequencing of the cloned amplicon. (C) The epitope of ZIKV EDIII recognized by the Z004-related antibody Z006 is shown in red (PDB: 5VIG). The E393-K394 residues are highlighted. (D) Impaired binding of Z004 to the EDIII escape mutants. ELISA demonstrates reduced binding of Z004 Fab to EDIII K394R and EDIII E393D . The positive control antibody Z015 recognizes an epitope that is independent of the E393 and K394 residues. Data are presented as mean ± SD of triplicates, and a representative of two experiments is shown. .

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, Sequencing, Clone Assay, Amplification, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control

3) Product Images from "Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR"

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Journal: Foods

doi: 10.3390/foods9030332

Gel electrophoresis of DNA extracted from different palm species to test the specificity of different sets of primer. Lane 1: Chilean wine palm ( Jubaea chilensis ); lane 2: oil palm ( Elaeis guineensis ); lane 3: coconut palm ( Cocos nucifera ), lane 4: jelly palm ( Butia capitata ); lane 5: non-template sample with water; lane 6: 100 bp DNA ladder. ( a ) PCR amplification with the primer set cocosPRK. ( b ) PCR amplification using the primer set cocosITS109. ( c ) PCR amplification performed with the primer set cocosITS197.
Figure Legend Snippet: Gel electrophoresis of DNA extracted from different palm species to test the specificity of different sets of primer. Lane 1: Chilean wine palm ( Jubaea chilensis ); lane 2: oil palm ( Elaeis guineensis ); lane 3: coconut palm ( Cocos nucifera ), lane 4: jelly palm ( Butia capitata ); lane 5: non-template sample with water; lane 6: 100 bp DNA ladder. ( a ) PCR amplification with the primer set cocosPRK. ( b ) PCR amplification using the primer set cocosITS109. ( c ) PCR amplification performed with the primer set cocosITS197.

Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification

Sensitivity test of the real-time PCR assay with 10-fold diluted DNA of coconut in a concentration range from 100 ng/µL to 0.1 pg/µL. The reactions were performed in duplicates. ( a ) The amplification plot shows C t values for samples from 100 ng/µL to 1 pg/µL. ( b ) Standard curve of the coconut TagMan real-time PCR system by plotting C t values against the log 10 input DNA concentration.
Figure Legend Snippet: Sensitivity test of the real-time PCR assay with 10-fold diluted DNA of coconut in a concentration range from 100 ng/µL to 0.1 pg/µL. The reactions were performed in duplicates. ( a ) The amplification plot shows C t values for samples from 100 ng/µL to 1 pg/µL. ( b ) Standard curve of the coconut TagMan real-time PCR system by plotting C t values against the log 10 input DNA concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Amplification

4) Product Images from "Purification of cross-linked RNA-protein complexes by phenol-toluol extraction"

Article Title: Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Journal: Nature Communications

doi: 10.1038/s41467-019-08942-3

PTex is a fast method to purify cross-linked RNPs. a In vivo cross-linking of HEK293 cells using UV light at 254 nm wavelength results in covalent bonds between RNA and proteins in direct contact. Cross-linked RNPs are indicated by an orange star. b Schematic of the separation principle of biphasic organic extractions used in PTex. Left panel: Phenol-Toluol (50:50) and neutral pH results in an accumulation of proteins and RNA in the upper aqueous phase (aq) while DNA and lipids are retained at the interphase (inter). Right panel: under acidic phenol and chaotropic conditions, non-cross-linked RNA accumulates in the aqueous phase (aq), non-cross-linked proteins in the lower organic phase (org), and cross-linked RNPs (clRNPs) are enriched at the interphase (inter). c Step-by-step analysis of proteins in 9 intermediary steps of the PTex protocol (3 extractions with 3 phases each). Western blot against HuR (ELAVL1, 35 kDa) demonstrates that UV-cross-linking-stabilised HuR-RNA complexes (upper edge/gel pocket of the blot) are largely enriched after PTex (step 3 interphase). A purified fly protein (Sxl RBD4) served as spike-in as 100% non-cross-linked RBP. d 5′-end radioactive-labelled RNA was subjected to PTex in vitro. e PCR with specific primers against exon 5 of the interleukin 3 (IL3) gene demonstrates efficient removal of genomic DNA after either full HEK293 cells (upper panel) or pre-purified genomic DNA (middle panel) were subjected to PTex. A PCR product derived from linear pUC19 DNA (lower panel) is also removed. f Enrichment of known RBPs by PTex tested by western-blot against PTBP1, FUS, or against non-classical RNA-binding enzymes Eno1 and GAPDH. Note that RNaseA treatment was performed after PTex as it removes partially shifted bands (smear) for some RBPs. g PTex enriches for cross-linked RBPs. RNase treatment before PTex strongly reduces recovery of known RNA-binders (PTBP1, FUS). Non-RBP controls Histone H3 and actin (ACTB) are efficiently depleted by PTex ( c , f , g ). For full gels/blots see Supplementary Figures 1 – 8
Figure Legend Snippet: PTex is a fast method to purify cross-linked RNPs. a In vivo cross-linking of HEK293 cells using UV light at 254 nm wavelength results in covalent bonds between RNA and proteins in direct contact. Cross-linked RNPs are indicated by an orange star. b Schematic of the separation principle of biphasic organic extractions used in PTex. Left panel: Phenol-Toluol (50:50) and neutral pH results in an accumulation of proteins and RNA in the upper aqueous phase (aq) while DNA and lipids are retained at the interphase (inter). Right panel: under acidic phenol and chaotropic conditions, non-cross-linked RNA accumulates in the aqueous phase (aq), non-cross-linked proteins in the lower organic phase (org), and cross-linked RNPs (clRNPs) are enriched at the interphase (inter). c Step-by-step analysis of proteins in 9 intermediary steps of the PTex protocol (3 extractions with 3 phases each). Western blot against HuR (ELAVL1, 35 kDa) demonstrates that UV-cross-linking-stabilised HuR-RNA complexes (upper edge/gel pocket of the blot) are largely enriched after PTex (step 3 interphase). A purified fly protein (Sxl RBD4) served as spike-in as 100% non-cross-linked RBP. d 5′-end radioactive-labelled RNA was subjected to PTex in vitro. e PCR with specific primers against exon 5 of the interleukin 3 (IL3) gene demonstrates efficient removal of genomic DNA after either full HEK293 cells (upper panel) or pre-purified genomic DNA (middle panel) were subjected to PTex. A PCR product derived from linear pUC19 DNA (lower panel) is also removed. f Enrichment of known RBPs by PTex tested by western-blot against PTBP1, FUS, or against non-classical RNA-binding enzymes Eno1 and GAPDH. Note that RNaseA treatment was performed after PTex as it removes partially shifted bands (smear) for some RBPs. g PTex enriches for cross-linked RBPs. RNase treatment before PTex strongly reduces recovery of known RNA-binders (PTBP1, FUS). Non-RBP controls Histone H3 and actin (ACTB) are efficiently depleted by PTex ( c , f , g ). For full gels/blots see Supplementary Figures 1 – 8

Techniques Used: In Vivo, Western Blot, Purification, In Vitro, Polymerase Chain Reaction, Derivative Assay, RNA Binding Assay

5) Product Images from "Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius"

Article Title: Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Journal: Archaea

doi: 10.1155/2017/7459310

Schematic of the multiple gene knockout system with one-step PCR (MONSTER). (a) Construction of a DNA photolyase-encoding gene ( phr ) deletion mutant. A plasmid-borne pyrE - lacS marker served as the PCR template, which attached S. acidocaldarius chromosomal sequences (5′, 3′, and partial sequences of phr at the 5′ ends of the primers) to the ends of the selectable dual marker. After one-step construction, the MONSTER-phr was electroporated into strain SK-1. A double crossover between the MONSTER-phr and the chromosome at the 5′ and Tg regions results in the pyrE - lacS marker and 3′ region insertion at the phr locus. The resulting uracil prototroph transformants that exhibit blue colonies can be selected on uracil-free plates. A DNA photolyase deletion mutant with the marker removed was generated by pop-out recombination at two duplicated 3′ regions, which can be selected by 5-FOA counterselection in combination with X-gal staining. Arrows show the positions of PCR primer sets. (b) Uracil and blue selection plate. (c) 5-FOA and white selection plate. (d) PCR analysis of the phr locus of the S. acidocaldarius strains SK-1 (Δ pyrE Δ suaI ), DP-1 Int (intermediate), and DP-1 (Δ pyrE Δ suaI Δ phr ) using phr-out-F/R as primers. The expected sizes of the PCR bands were 1.5 kb (wt), 4 kb (recombinant), and 0.2 kb (deletion mutant). A λ -EcoT14 or 100 bp DNA ladder was loaded in lane M.
Figure Legend Snippet: Schematic of the multiple gene knockout system with one-step PCR (MONSTER). (a) Construction of a DNA photolyase-encoding gene ( phr ) deletion mutant. A plasmid-borne pyrE - lacS marker served as the PCR template, which attached S. acidocaldarius chromosomal sequences (5′, 3′, and partial sequences of phr at the 5′ ends of the primers) to the ends of the selectable dual marker. After one-step construction, the MONSTER-phr was electroporated into strain SK-1. A double crossover between the MONSTER-phr and the chromosome at the 5′ and Tg regions results in the pyrE - lacS marker and 3′ region insertion at the phr locus. The resulting uracil prototroph transformants that exhibit blue colonies can be selected on uracil-free plates. A DNA photolyase deletion mutant with the marker removed was generated by pop-out recombination at two duplicated 3′ regions, which can be selected by 5-FOA counterselection in combination with X-gal staining. Arrows show the positions of PCR primer sets. (b) Uracil and blue selection plate. (c) 5-FOA and white selection plate. (d) PCR analysis of the phr locus of the S. acidocaldarius strains SK-1 (Δ pyrE Δ suaI ), DP-1 Int (intermediate), and DP-1 (Δ pyrE Δ suaI Δ phr ) using phr-out-F/R as primers. The expected sizes of the PCR bands were 1.5 kb (wt), 4 kb (recombinant), and 0.2 kb (deletion mutant). A λ -EcoT14 or 100 bp DNA ladder was loaded in lane M.

Techniques Used: Gene Knockout, Polymerase Chain Reaction, Mutagenesis, Plasmid Preparation, Marker, Generated, Staining, Selection, Recombinant

In-frame deletion of argD via the MONSTER. (a) Construction of an argD deletion mutant. A plasmid-borne pyrE - lacS marker served as the PCR template, which attached S. acidocaldarius chromosomal sequences (5′, 3′, and partial sequences of argD at the 5′ ends of the primers) to the ends of the selectable dual marker. After one-step construction, the MONSTER-argD was electroporated into strain SK-1. A double crossover between the MONSTER-argD and the chromosome at the Tg and 3′ regions results in the pyrE - lacS marker and 5′ region insertion at the argD locus. The resulting uracil prototroph transformants exhibit blue colonies and can be selected on uracil-free plates. An argD deletion mutant with the marker removed was generated by pop-out recombination at two duplicated 5′ regions, which can be selected by 5-FOA counterselection in combination with X-gal staining. Arrows show the positions of PCR primer sets. (b) Uracil and blue selection plate. (c) 5-FOA and white selection plate. (d) PCR analysis of the argD locus of the S. acidocaldarius strains SK-1 (Δ pyrE Δ suaI ), SK-5 Int (intermediate), and SK-5 (Δ pyrE Δ suaI Δ argD ) using argD-F-F/R as primers. The expected sizes of the PCR bands were 0.5 kb (wt), 3 kb (recombinant), and 0.1 kb (deletion mutant). A λ -EcoT14 or 100 bp DNA ladder was loaded in lane M.
Figure Legend Snippet: In-frame deletion of argD via the MONSTER. (a) Construction of an argD deletion mutant. A plasmid-borne pyrE - lacS marker served as the PCR template, which attached S. acidocaldarius chromosomal sequences (5′, 3′, and partial sequences of argD at the 5′ ends of the primers) to the ends of the selectable dual marker. After one-step construction, the MONSTER-argD was electroporated into strain SK-1. A double crossover between the MONSTER-argD and the chromosome at the Tg and 3′ regions results in the pyrE - lacS marker and 5′ region insertion at the argD locus. The resulting uracil prototroph transformants exhibit blue colonies and can be selected on uracil-free plates. An argD deletion mutant with the marker removed was generated by pop-out recombination at two duplicated 5′ regions, which can be selected by 5-FOA counterselection in combination with X-gal staining. Arrows show the positions of PCR primer sets. (b) Uracil and blue selection plate. (c) 5-FOA and white selection plate. (d) PCR analysis of the argD locus of the S. acidocaldarius strains SK-1 (Δ pyrE Δ suaI ), SK-5 Int (intermediate), and SK-5 (Δ pyrE Δ suaI Δ argD ) using argD-F-F/R as primers. The expected sizes of the PCR bands were 0.5 kb (wt), 3 kb (recombinant), and 0.1 kb (deletion mutant). A λ -EcoT14 or 100 bp DNA ladder was loaded in lane M.

Techniques Used: Mutagenesis, Plasmid Preparation, Marker, Polymerase Chain Reaction, Generated, Staining, Selection, Recombinant

6) Product Images from "Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells"

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0175585

Preparation of repeat DNA and construction of plasmids. (A to D) Structure of the plasmids used in this study. (E and F) Direct or inverted repeat DNA was prepared by PCR amplification, digestion with Rsr II (R) or double digestion with Sal I (S) and Mlu 1 (M), respectively, followed by ligation. (G to I) G5 (G), G5AR1 (H), or a portion of λ (I) DNA before (lanes 1 and 3) and after ligation to direct repeats (lane 2) or inverted repeats (lane 4). DNA was separated by agarose gel electrophoresis. M; molecular weight marker.
Figure Legend Snippet: Preparation of repeat DNA and construction of plasmids. (A to D) Structure of the plasmids used in this study. (E and F) Direct or inverted repeat DNA was prepared by PCR amplification, digestion with Rsr II (R) or double digestion with Sal I (S) and Mlu 1 (M), respectively, followed by ligation. (G to I) G5 (G), G5AR1 (H), or a portion of λ (I) DNA before (lanes 1 and 3) and after ligation to direct repeats (lane 2) or inverted repeats (lane 4). DNA was separated by agarose gel electrophoresis. M; molecular weight marker.

Techniques Used: Polymerase Chain Reaction, Amplification, Ligation, Agarose Gel Electrophoresis, Molecular Weight, Marker

Copy number of the transfected sequence in the stable transformants. After transfection of the indicated DNA into COLO 320DM or CHO DG44 cells, the stable transformants were selected by blasticidin for 1 month. The genomic DNA was isolated and subjected to real-time PCR to determine the amount of SRα promoter, G5, λ-phage, and Gapdh sequence. The PCR reaction was done in triplicate, and the mean value was used to calculate the copy number of each sequence relative to Gapdh (see graph). The deviation between triplicate reactions was too small to be represented with error bars.
Figure Legend Snippet: Copy number of the transfected sequence in the stable transformants. After transfection of the indicated DNA into COLO 320DM or CHO DG44 cells, the stable transformants were selected by blasticidin for 1 month. The genomic DNA was isolated and subjected to real-time PCR to determine the amount of SRα promoter, G5, λ-phage, and Gapdh sequence. The PCR reaction was done in triplicate, and the mean value was used to calculate the copy number of each sequence relative to Gapdh (see graph). The deviation between triplicate reactions was too small to be represented with error bars.

Techniques Used: Transfection, Sequencing, Isolation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

7) Product Images from "Circulating immune cells mediate a systemic RNAi based adaptive antiviral response in Drosophila"

Article Title: Circulating immune cells mediate a systemic RNAi based adaptive antiviral response in Drosophila

Journal: Cell

doi: 10.1016/j.cell.2017.03.033

Stable viral cDNA (vDNA) production in haemocytes is an Ago2 and reverse transcriptase-dependent process, involved in vsRNA de novo synthesis (A) Experimental design to identify vDNA production in haemocytes and its role in vsRNA de novo synthesis. (B) Production of vDNA in haemocytes. DNA was extracted from haemocyte-enriched fraction (Hem) or carcasses (Carc) of naïve (lanes 1, 2) or SINV-infected flies (lanes 3 to 11), treated with RNase A then assessed for SINV DNA product by end-point PCR. Crq gene was used as loading control. w 1118 flies were used in lanes 1 to 8. (C) Inhibition of vDNA production by AZT injection in flies reduces the production of primary (pRNA-seq) and secondary (tripRNA-seq) vsRNAs. Flies were injected with 2.5 nmoles AZT (red) or PBS only (grey) 1 day before and 1 day after SINV injection. Small RNAs were extracted at 4 d.p.i and subjected in parallel to pRNA-seq and tripRNA-seq. vsRNA counts given per 10 4 SINV genomes. (D) AZT treatment increases SINV replication in an Ago2-dependent manner. Absolute SINV titers measured by RT-qPCR in wild type ( w 1118 ) or ago2 mutant ( ago2 414 ) flies treated with AZT (as in C) (n=3 injection experiments). (E) vDNAs are retained specifically in haemocytes as a form of long lasting immunological memory. 3 weeks after SINV infection (+SINV), the haemocyte-enriched fraction (Hem) was isolated from flies expressing eGFP in their haemocytes ( hml > eGFP ). Half of the fraction was further processed by FACS sorting to obtain pure haemocytes samples (Hem FACS). Both preparations were treated with RNase A then assessed for SINV DNA product by end-point PCR. Crq gene was used as loading control. (F) Model for the interactions between Ago2, reverse-transcriptase (endoRT), vDNA and vsRNA synthesis in haemocytes.
Figure Legend Snippet: Stable viral cDNA (vDNA) production in haemocytes is an Ago2 and reverse transcriptase-dependent process, involved in vsRNA de novo synthesis (A) Experimental design to identify vDNA production in haemocytes and its role in vsRNA de novo synthesis. (B) Production of vDNA in haemocytes. DNA was extracted from haemocyte-enriched fraction (Hem) or carcasses (Carc) of naïve (lanes 1, 2) or SINV-infected flies (lanes 3 to 11), treated with RNase A then assessed for SINV DNA product by end-point PCR. Crq gene was used as loading control. w 1118 flies were used in lanes 1 to 8. (C) Inhibition of vDNA production by AZT injection in flies reduces the production of primary (pRNA-seq) and secondary (tripRNA-seq) vsRNAs. Flies were injected with 2.5 nmoles AZT (red) or PBS only (grey) 1 day before and 1 day after SINV injection. Small RNAs were extracted at 4 d.p.i and subjected in parallel to pRNA-seq and tripRNA-seq. vsRNA counts given per 10 4 SINV genomes. (D) AZT treatment increases SINV replication in an Ago2-dependent manner. Absolute SINV titers measured by RT-qPCR in wild type ( w 1118 ) or ago2 mutant ( ago2 414 ) flies treated with AZT (as in C) (n=3 injection experiments). (E) vDNAs are retained specifically in haemocytes as a form of long lasting immunological memory. 3 weeks after SINV infection (+SINV), the haemocyte-enriched fraction (Hem) was isolated from flies expressing eGFP in their haemocytes ( hml > eGFP ). Half of the fraction was further processed by FACS sorting to obtain pure haemocytes samples (Hem FACS). Both preparations were treated with RNase A then assessed for SINV DNA product by end-point PCR. Crq gene was used as loading control. (F) Model for the interactions between Ago2, reverse-transcriptase (endoRT), vDNA and vsRNA synthesis in haemocytes.

Techniques Used: Infection, Polymerase Chain Reaction, Inhibition, Injection, Quantitative RT-PCR, Mutagenesis, Isolation, Expressing, FACS

Uptake of exogenous dsRNA by haemocytes stimulates vDNA production (A) Haemocytes can efficiently take up dsRNA to silence an endogenously expressed reporter gene (eGFP) in vivo . 50 ng of control dsRNA (dsCTR) or anti-eGFP dsRNA (dsGFP) were injected in flies expressing eGFP in the haemocytes ( hml > eGFP ). eGFP expression was monitored directly by fluorescence microscopy at 5 days post injection (d.p.i.) ( i. top panel) or by western blot at 0, 5, 10 and 20 d.p.i.( ii. lower panel). (B) Loss of RNAi-competent haemocytes inhibits antiviral protection induced by dsRNA immunization. Control (dsCTR) or anti-SINV (dsSIN) dsRNA were co-injected with 500 pfu SINV in control flies ( hml > dsCtr ), haemocyte-depleted flies ( hml > reaper ) or flies with RNAi-deficient haemocytes ( hml > dsAgo2). dsSIN protection efficiency was measured by RT-qPCR as the ratio of SINV titers in dsSIN versus dsCTR treated flies at 3 and 4 d.p.i (n = 3 injection experiments). (C) anti-SINV dsRNA injection increases production of SINV vDNA in haemocytes. SINV vDNA production was assessed by end point PCR against SINV NSP1 gene (insert) and by qPCR in haemocytes of SINV infected flies co-injected with control (dsCtr) or anti-SINV (dsSIN) dsRNA. qPCR analysis shows that vDNA production from genes in both the viral genomic region (NSP1, in red) or the viral subgenomic region (capsid, in grey) was equally stimulated by dsSIN injection. (D) Model for stimulation of vDNA formation in haemocytes by exogenous dsRNA. Uptake of viral genomic RNA (single stranded and/or double-stranded RNA) in haemocytes stimulates vDNA production which allows increased de novo synthesis of vsRNAs and efficient systemic antiviral RNAi response.
Figure Legend Snippet: Uptake of exogenous dsRNA by haemocytes stimulates vDNA production (A) Haemocytes can efficiently take up dsRNA to silence an endogenously expressed reporter gene (eGFP) in vivo . 50 ng of control dsRNA (dsCTR) or anti-eGFP dsRNA (dsGFP) were injected in flies expressing eGFP in the haemocytes ( hml > eGFP ). eGFP expression was monitored directly by fluorescence microscopy at 5 days post injection (d.p.i.) ( i. top panel) or by western blot at 0, 5, 10 and 20 d.p.i.( ii. lower panel). (B) Loss of RNAi-competent haemocytes inhibits antiviral protection induced by dsRNA immunization. Control (dsCTR) or anti-SINV (dsSIN) dsRNA were co-injected with 500 pfu SINV in control flies ( hml > dsCtr ), haemocyte-depleted flies ( hml > reaper ) or flies with RNAi-deficient haemocytes ( hml > dsAgo2). dsSIN protection efficiency was measured by RT-qPCR as the ratio of SINV titers in dsSIN versus dsCTR treated flies at 3 and 4 d.p.i (n = 3 injection experiments). (C) anti-SINV dsRNA injection increases production of SINV vDNA in haemocytes. SINV vDNA production was assessed by end point PCR against SINV NSP1 gene (insert) and by qPCR in haemocytes of SINV infected flies co-injected with control (dsCtr) or anti-SINV (dsSIN) dsRNA. qPCR analysis shows that vDNA production from genes in both the viral genomic region (NSP1, in red) or the viral subgenomic region (capsid, in grey) was equally stimulated by dsSIN injection. (D) Model for stimulation of vDNA formation in haemocytes by exogenous dsRNA. Uptake of viral genomic RNA (single stranded and/or double-stranded RNA) in haemocytes stimulates vDNA production which allows increased de novo synthesis of vsRNAs and efficient systemic antiviral RNAi response.

Techniques Used: In Vivo, Injection, Expressing, Fluorescence, Microscopy, Western Blot, Quantitative RT-PCR, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Infection

8) Product Images from "Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform"

Article Title: Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform

Journal: Scientific Reports

doi: 10.1038/srep22259

Isolation and genome amplification of bacteria exhibiting BGL activities from surface and deep seawater. ( a ) Bright-field ( left ) and fluorescence ( right ) images of W/O microdroplets encapsulating environmental bacteria with FDGlu. The white arrowhead shows a fluorescent bacterial cell in a W/O microdroplet. Scale bar represents 20 μm. ( b ) PCR amplification of 16S rRNA genes from MDA products. The amplicons were analysed with 1% agarose gel electrophoresis and stained with SYBR Safe. The estimated amplicon size is approximately 1,466 bp. Lane M, DNA marker (λ- Eco T14 I digest); lane 1–4, MDA products from surface seawater (Droplet No. 1–4), lane 5–9, MDA products from deep seawater (Droplet No. 5–9).
Figure Legend Snippet: Isolation and genome amplification of bacteria exhibiting BGL activities from surface and deep seawater. ( a ) Bright-field ( left ) and fluorescence ( right ) images of W/O microdroplets encapsulating environmental bacteria with FDGlu. The white arrowhead shows a fluorescent bacterial cell in a W/O microdroplet. Scale bar represents 20 μm. ( b ) PCR amplification of 16S rRNA genes from MDA products. The amplicons were analysed with 1% agarose gel electrophoresis and stained with SYBR Safe. The estimated amplicon size is approximately 1,466 bp. Lane M, DNA marker (λ- Eco T14 I digest); lane 1–4, MDA products from surface seawater (Droplet No. 1–4), lane 5–9, MDA products from deep seawater (Droplet No. 5–9).

Techniques Used: Isolation, Amplification, Fluorescence, Polymerase Chain Reaction, Multiple Displacement Amplification, Agarose Gel Electrophoresis, Staining, Marker

9) Product Images from "In vivo targeted single-nucleotide editing in zebrafish"

Article Title: In vivo targeted single-nucleotide editing in zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-018-29794-9

Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
Figure Legend Snippet: Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

Techniques Used: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing

10) Product Images from "Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy"

Article Title: Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

Journal: Molecular Therapy

doi: 10.1038/mt.2016.92

Antisense oligonucleotides (AON) can induce skipping of exon 13 and 105 in dermal fibroblast . RT-PCR on RNA extracted from dermal fibroblast treated with 250 or 500 nmol/l of AONs designed against exon 13 ( a ) or 105 ( c ) shows efficient skipping of the respective exons. For both exons amplification of the region surrounding exon 13 (exon 12 to 14) or exon 105 (exon 102 to 106) reveals an additional lower band corresponding to an amplicon lacking exon 13 (144 bp) or 105 (81 pb), respectively. Sanger sequencing then confirms the precise skipping of exon 13 ( b ) or 105 ( d ). White asterisk indicates heteroduplex DNA.
Figure Legend Snippet: Antisense oligonucleotides (AON) can induce skipping of exon 13 and 105 in dermal fibroblast . RT-PCR on RNA extracted from dermal fibroblast treated with 250 or 500 nmol/l of AONs designed against exon 13 ( a ) or 105 ( c ) shows efficient skipping of the respective exons. For both exons amplification of the region surrounding exon 13 (exon 12 to 14) or exon 105 (exon 102 to 106) reveals an additional lower band corresponding to an amplicon lacking exon 13 (144 bp) or 105 (81 pb), respectively. Sanger sequencing then confirms the precise skipping of exon 13 ( b ) or 105 ( d ). White asterisk indicates heteroduplex DNA.

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

Related Articles

Transfection:

Article Title: Circulating immune cells mediate a systemic RNAi based adaptive antiviral response in Drosophila
Article Snippet: .. After DNA clean up (Nucleospin Gel and PCR clean-up, Macherey-Nagel), capped viral RNA was synthesized using mMESSAGE SP6 kit (Ambion) and transfected in BHK cells. .. P0 viral stock was collected at 24 hours post transfection and propagated in BHK cells to generated the P1 stock at 24 hours post infection.

Amplification:

Article Title: A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus Escape Mutations in Non-human Primates
Article Snippet: .. RNA was extracted from TRIzol-LS (Life Technologies) and reverse transcribed with Superscript III RT (Thermo Fisher Scientific) and random primers according to the company’s protocol prior to PCR amplification of the ZIKV EDIII region with primers DFRp1284 and DFRp1469 followed by PCR clean-up with Nucleospin (Macherey-Nagel) and direct sequencing with primer DFRp1283. .. Where necessary, a second round of nested PCR was performed with primers DFRp1472 and DFRp1470.

Agarose Gel Electrophoresis:

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR
Article Snippet: .. DNA Sequencing To confirm the sequence of PCR products, the DNA was isolated from the agarose gel using a PCR clean-up and gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and was sent to a laboratory specialized in sequencing (Eurofins Genomics Germany GmbH, Cologne, Germany). .. The results were verified using a sequence comparison with BLASTn against the NCBI nucleotide database.

Synthesized:

Article Title: Circulating immune cells mediate a systemic RNAi based adaptive antiviral response in Drosophila
Article Snippet: .. After DNA clean up (Nucleospin Gel and PCR clean-up, Macherey-Nagel), capped viral RNA was synthesized using mMESSAGE SP6 kit (Ambion) and transfected in BHK cells. .. P0 viral stock was collected at 24 hours post transfection and propagated in BHK cells to generated the P1 stock at 24 hours post infection.

Isolation:

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR
Article Snippet: .. DNA Sequencing To confirm the sequence of PCR products, the DNA was isolated from the agarose gel using a PCR clean-up and gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and was sent to a laboratory specialized in sequencing (Eurofins Genomics Germany GmbH, Cologne, Germany). .. The results were verified using a sequence comparison with BLASTn against the NCBI nucleotide database.

Ligation:

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells
Article Snippet: .. The DNA was purified using NucleoSpin® Gel and PCR Clean-up (MACHEREY-NAGEL Co.) and ligated using Ligation high Ver.2 (TOYOBO Co.). .. The ligated DNA was purified by phenol/chloroform extraction and ethanol precipitation, and was used for cell transfection.

Purification:

Article Title: Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform
Article Snippet: .. The amplicons were purified (NucleoSpin Gel and PCR Clean-up, Macherey-Nagel GmbH & Co. KG) and subjected to direct sequencing by the Sanger method. ..

Article Title: Purification of cross-linked RNA-protein complexes by phenol-toluol extraction
Article Snippet: .. Plasmids were purified using the NucleoBond Xtra Midi kit (Macherey-Nagel, 740410.100), DNA fragments by NucleoSpin Gel and PCR clean-up (Macherey-Nagel, 740609.50) and RNA by acidic phenol extraction . .. RNA 5′-GAG UUU UUU UAC A-3′ (13 nt) was synthesised by Biomers (Ulm, Germany).

Article Title: Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius
Article Snippet: .. PCR products and plasmid DNA were purified using NucleoSpin Gel and PCR Clean-up and NucleoSpin QuickPure kits (Macherey-Nagel), respectively. .. Construction of Marker Cassettes The plasmid and linear DNA used in this study are shown in and the PCR primers used are listed in .

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells
Article Snippet: .. The DNA was purified using NucleoSpin® Gel and PCR Clean-up (MACHEREY-NAGEL Co.) and ligated using Ligation high Ver.2 (TOYOBO Co.). .. The ligated DNA was purified by phenol/chloroform extraction and ethanol precipitation, and was used for cell transfection.

Article Title: In vivo targeted single-nucleotide editing in zebrafish
Article Snippet: .. The PCR products from the chd and oep amplifications were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced by Fasmac (Atsugi, Japan) or by Macrogen Japan (Kyoto, Japan) with the chd _1st_F and oep _1st_F primers, respectively. .. Primers used in allele-specific PCR are listed in Suppl.

Polymerase Chain Reaction:

Article Title: Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform
Article Snippet: .. The amplicons were purified (NucleoSpin Gel and PCR Clean-up, Macherey-Nagel GmbH & Co. KG) and subjected to direct sequencing by the Sanger method. ..

Article Title: Purification of cross-linked RNA-protein complexes by phenol-toluol extraction
Article Snippet: .. Plasmids were purified using the NucleoBond Xtra Midi kit (Macherey-Nagel, 740410.100), DNA fragments by NucleoSpin Gel and PCR clean-up (Macherey-Nagel, 740609.50) and RNA by acidic phenol extraction . .. RNA 5′-GAG UUU UUU UAC A-3′ (13 nt) was synthesised by Biomers (Ulm, Germany).

Article Title: Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius
Article Snippet: .. PCR products and plasmid DNA were purified using NucleoSpin Gel and PCR Clean-up and NucleoSpin QuickPure kits (Macherey-Nagel), respectively. .. Construction of Marker Cassettes The plasmid and linear DNA used in this study are shown in and the PCR primers used are listed in .

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells
Article Snippet: .. The DNA was purified using NucleoSpin® Gel and PCR Clean-up (MACHEREY-NAGEL Co.) and ligated using Ligation high Ver.2 (TOYOBO Co.). .. The ligated DNA was purified by phenol/chloroform extraction and ethanol precipitation, and was used for cell transfection.

Article Title: A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus Escape Mutations in Non-human Primates
Article Snippet: .. RNA was extracted from TRIzol-LS (Life Technologies) and reverse transcribed with Superscript III RT (Thermo Fisher Scientific) and random primers according to the company’s protocol prior to PCR amplification of the ZIKV EDIII region with primers DFRp1284 and DFRp1469 followed by PCR clean-up with Nucleospin (Macherey-Nagel) and direct sequencing with primer DFRp1283. .. Where necessary, a second round of nested PCR was performed with primers DFRp1472 and DFRp1470.

Article Title: Circulating immune cells mediate a systemic RNAi based adaptive antiviral response in Drosophila
Article Snippet: .. After DNA clean up (Nucleospin Gel and PCR clean-up, Macherey-Nagel), capped viral RNA was synthesized using mMESSAGE SP6 kit (Ambion) and transfected in BHK cells. .. P0 viral stock was collected at 24 hours post transfection and propagated in BHK cells to generated the P1 stock at 24 hours post infection.

Article Title: In vivo targeted single-nucleotide editing in zebrafish
Article Snippet: .. The PCR products from the chd and oep amplifications were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced by Fasmac (Atsugi, Japan) or by Macrogen Japan (Kyoto, Japan) with the chd _1st_F and oep _1st_F primers, respectively. .. Primers used in allele-specific PCR are listed in Suppl.

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR
Article Snippet: .. DNA Sequencing To confirm the sequence of PCR products, the DNA was isolated from the agarose gel using a PCR clean-up and gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and was sent to a laboratory specialized in sequencing (Eurofins Genomics Germany GmbH, Cologne, Germany). .. The results were verified using a sequence comparison with BLASTn against the NCBI nucleotide database.

DNA Sequencing:

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR
Article Snippet: .. DNA Sequencing To confirm the sequence of PCR products, the DNA was isolated from the agarose gel using a PCR clean-up and gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and was sent to a laboratory specialized in sequencing (Eurofins Genomics Germany GmbH, Cologne, Germany). .. The results were verified using a sequence comparison with BLASTn against the NCBI nucleotide database.

Sequencing:

Article Title: Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform
Article Snippet: .. The amplicons were purified (NucleoSpin Gel and PCR Clean-up, Macherey-Nagel GmbH & Co. KG) and subjected to direct sequencing by the Sanger method. ..

Article Title: A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus Escape Mutations in Non-human Primates
Article Snippet: .. RNA was extracted from TRIzol-LS (Life Technologies) and reverse transcribed with Superscript III RT (Thermo Fisher Scientific) and random primers according to the company’s protocol prior to PCR amplification of the ZIKV EDIII region with primers DFRp1284 and DFRp1469 followed by PCR clean-up with Nucleospin (Macherey-Nagel) and direct sequencing with primer DFRp1283. .. Where necessary, a second round of nested PCR was performed with primers DFRp1472 and DFRp1470.

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR
Article Snippet: .. DNA Sequencing To confirm the sequence of PCR products, the DNA was isolated from the agarose gel using a PCR clean-up and gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and was sent to a laboratory specialized in sequencing (Eurofins Genomics Germany GmbH, Cologne, Germany). .. The results were verified using a sequence comparison with BLASTn against the NCBI nucleotide database.

Gel Extraction:

Article Title: Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR
Article Snippet: .. DNA Sequencing To confirm the sequence of PCR products, the DNA was isolated from the agarose gel using a PCR clean-up and gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and was sent to a laboratory specialized in sequencing (Eurofins Genomics Germany GmbH, Cologne, Germany). .. The results were verified using a sequence comparison with BLASTn against the NCBI nucleotide database.

Plasmid Preparation:

Article Title: Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius
Article Snippet: .. PCR products and plasmid DNA were purified using NucleoSpin Gel and PCR Clean-up and NucleoSpin QuickPure kits (Macherey-Nagel), respectively. .. Construction of Marker Cassettes The plasmid and linear DNA used in this study are shown in and the PCR primers used are listed in .

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  • 94
    MACHEREY NAGEL pcr clean up kit
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
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    Real-time <t>PCR</t> analysis shows loss of endonuclease expression and associated loss of editing. Relative RNA abundance is shown for KREN1, KREN2, KREN3 and never-edited mRNAs COI and ND4 (black bars), pre-edited mRNAs (white bars), and edited mRNAs (gray bars). For each target <t>amplicon,</t> the relative change in RNA abundance was determined by using telomerase reverse transcriptase (TERT) mRNA as an internal control, with each cell line compared relative to either 427wt or MGA control as indicated. ( A ) MGA cells have no large changes in mRNA abundance relative to 427wt, with the exception of the complete loss of CYb editing. As the remaining cell lines are compared to MGA, CYb is excluded from those analyses. ( B ) KREN1 only cells have no detectable KREN2 or KREN3 mRNA, and a broad loss of RNA editing. ( C ) KREN2 only cells have no detectable KREN1 or KREN3 mRNA, and a broad loss of RNA editing. ( D ) KREN3 only cells have no detectable KREN1 or KREN2 mRNA, and a broad loss of RNA editing, with COII editing notably retained. ( E ) Triple null cells have no detectable KREN1, KREN2, or KREN3 mRNA, and a broad loss of RNA editing. ( F ) KREN2 null cells have no detectable KREN2 mRNA, and a broad loss of RNA editing with some amount of COII and ND7 editing retained. ( G ) KREN3 null cells have no detectable KREN3 mRNA, and a loss of COII RNA editing with variable amounts of other edited mRNAs retained.
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    MACHEREY NAGEL pcr clean up
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
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    MACHEREY NAGEL pcr clean up columns
    Characterization of a stable <t>eys</t> rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of <t>RT-PCR</t> analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
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    Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Journal: Viruses

    Article Title: A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

    doi: 10.3390/v11121129

    Figure Lengend Snippet: Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Article Snippet: Amplified DNA Clean Up REPLI-g samples were purified using the NucleoSpin Gel and PCR clean-up Kit (Macherey-Nagel Düren, Germany).

    Techniques: Polymerase Chain Reaction, Whole Genome Amplification

    Real-time PCR analysis shows loss of endonuclease expression and associated loss of editing. Relative RNA abundance is shown for KREN1, KREN2, KREN3 and never-edited mRNAs COI and ND4 (black bars), pre-edited mRNAs (white bars), and edited mRNAs (gray bars). For each target amplicon, the relative change in RNA abundance was determined by using telomerase reverse transcriptase (TERT) mRNA as an internal control, with each cell line compared relative to either 427wt or MGA control as indicated. ( A ) MGA cells have no large changes in mRNA abundance relative to 427wt, with the exception of the complete loss of CYb editing. As the remaining cell lines are compared to MGA, CYb is excluded from those analyses. ( B ) KREN1 only cells have no detectable KREN2 or KREN3 mRNA, and a broad loss of RNA editing. ( C ) KREN2 only cells have no detectable KREN1 or KREN3 mRNA, and a broad loss of RNA editing. ( D ) KREN3 only cells have no detectable KREN1 or KREN2 mRNA, and a broad loss of RNA editing, with COII editing notably retained. ( E ) Triple null cells have no detectable KREN1, KREN2, or KREN3 mRNA, and a broad loss of RNA editing. ( F ) KREN2 null cells have no detectable KREN2 mRNA, and a broad loss of RNA editing with some amount of COII and ND7 editing retained. ( G ) KREN3 null cells have no detectable KREN3 mRNA, and a loss of COII RNA editing with variable amounts of other edited mRNAs retained.

    Journal: Nucleic Acids Research

    Article Title: In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases

    doi: 10.1093/nar/gkx116

    Figure Lengend Snippet: Real-time PCR analysis shows loss of endonuclease expression and associated loss of editing. Relative RNA abundance is shown for KREN1, KREN2, KREN3 and never-edited mRNAs COI and ND4 (black bars), pre-edited mRNAs (white bars), and edited mRNAs (gray bars). For each target amplicon, the relative change in RNA abundance was determined by using telomerase reverse transcriptase (TERT) mRNA as an internal control, with each cell line compared relative to either 427wt or MGA control as indicated. ( A ) MGA cells have no large changes in mRNA abundance relative to 427wt, with the exception of the complete loss of CYb editing. As the remaining cell lines are compared to MGA, CYb is excluded from those analyses. ( B ) KREN1 only cells have no detectable KREN2 or KREN3 mRNA, and a broad loss of RNA editing. ( C ) KREN2 only cells have no detectable KREN1 or KREN3 mRNA, and a broad loss of RNA editing. ( D ) KREN3 only cells have no detectable KREN1 or KREN2 mRNA, and a broad loss of RNA editing, with COII editing notably retained. ( E ) Triple null cells have no detectable KREN1, KREN2, or KREN3 mRNA, and a broad loss of RNA editing. ( F ) KREN2 null cells have no detectable KREN2 mRNA, and a broad loss of RNA editing with some amount of COII and ND7 editing retained. ( G ) KREN3 null cells have no detectable KREN3 mRNA, and a loss of COII RNA editing with variable amounts of other edited mRNAs retained.

    Article Snippet: PCR amplicon DNA was isolated from gel slices using NuceloSpin Gel and PCR Clean-up spin columns, eluting in 30 μl volume (Macherey-Nagel).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Amplification

    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Journal: Scientific Reports

    Article Title: In vivo targeted single-nucleotide editing in zebrafish

    doi: 10.1038/s41598-018-29794-9

    Figure Lengend Snippet: Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Article Snippet: The PCR products from the chd and oep amplifications were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced by Fasmac (Atsugi, Japan) or by Macrogen Japan (Kyoto, Japan) with the chd _1st_F and oep _1st_F primers, respectively.

    Techniques: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing

    Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Journal: PLoS ONE

    Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

    doi: 10.1371/journal.pone.0200789

    Figure Lengend Snippet: Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Article Snippet: To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction