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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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1) Product Images from "A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus"

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

Journal: Viruses

doi: 10.3390/v11121129

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.
Figure Legend 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.

Techniques Used: Polymerase Chain Reaction, Whole Genome Amplification

2) Product Images from "Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita"

Article Title: Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita

Journal: Scientific Reports

doi: 10.1038/s41598-018-37321-z

Relative transcript levels of QQ-ORFs in A . aurita polyps and ephyrae. Relative transcript levels of aaqq1 , aaqq2 and aaqq3 were determined by qRT-PCR analysis in A . aurita polyps and ephyrae with at least three independent biological experiments, each with three technical replicates. ( A ) Polyps and ephyrae were kept under native and germ-free conditions. ( B–E ) Polyps and ephyrae were kept under native ( B+D ) and germ-free ( C+E ) conditions, additionally challenged with selected bacteria, K . oxytoca , V . parahaemolyticus and P . tunicata . Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene actin .
Figure Legend Snippet: Relative transcript levels of QQ-ORFs in A . aurita polyps and ephyrae. Relative transcript levels of aaqq1 , aaqq2 and aaqq3 were determined by qRT-PCR analysis in A . aurita polyps and ephyrae with at least three independent biological experiments, each with three technical replicates. ( A ) Polyps and ephyrae were kept under native and germ-free conditions. ( B–E ) Polyps and ephyrae were kept under native ( B+D ) and germ-free ( C+E ) conditions, additionally challenged with selected bacteria, K . oxytoca , V . parahaemolyticus and P . tunicata . Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene actin .

Techniques Used: Quantitative RT-PCR

Characterization of A . aurita QQ ORFs. ( A ) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1 , aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). ( B ) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. ( C ) QQ proteins were analyzed using secondary structure prediction software PredictProtein ( http://www.predictprotein.org/ ) with following visual output in Profsec: yellow, strand; red, helix. InterProScan ( http://www.ebi.ac.uk/Tools/pfa/iprscan/ ) and GlobPlot ( http://globplot.embl.de/ ) were used for functional protein analysis. ( D ) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.
Figure Legend Snippet: Characterization of A . aurita QQ ORFs. ( A ) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1 , aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). ( B ) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. ( C ) QQ proteins were analyzed using secondary structure prediction software PredictProtein ( http://www.predictprotein.org/ ) with following visual output in Profsec: yellow, strand; red, helix. InterProScan ( http://www.ebi.ac.uk/Tools/pfa/iprscan/ ) and GlobPlot ( http://globplot.embl.de/ ) were used for functional protein analysis. ( D ) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.

Techniques Used: Clone Assay, Purification, Affinity Chromatography, SDS Page, Software, Functional Assay, Polymerase Chain Reaction, Amplification

3) Product Images from "FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli"

Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0129547

Construction of FabV and FabI-containing plasmid vectors. The bla gene (AmpR) was removed and restriction enzyme ( Cla I and Sma I) sites were introduced downstream P3 promoter of β-lactamase by whole-plasmid PCR of pUC19, pSA-HP24, and pBR322 vectors. Inserts were prepared by PCR amplifying fab V and fab I using Vibrio cholerae O1 El Tor or E . coli BL21(DE3) genomic DNA respectively as DNA template. Vectors and inserts were restricted using Cla I and Sma I, gel purified, and ligated at 1:3 vector/insert ratio. These manipulations resulted in the construction of (A) FabV-containing pUC19, (B) FabI-containing pUC19, (C) FabV-containing pSA-HP24, and (D) FabI-containing pBR322 plasmid vectors.
Figure Legend Snippet: Construction of FabV and FabI-containing plasmid vectors. The bla gene (AmpR) was removed and restriction enzyme ( Cla I and Sma I) sites were introduced downstream P3 promoter of β-lactamase by whole-plasmid PCR of pUC19, pSA-HP24, and pBR322 vectors. Inserts were prepared by PCR amplifying fab V and fab I using Vibrio cholerae O1 El Tor or E . coli BL21(DE3) genomic DNA respectively as DNA template. Vectors and inserts were restricted using Cla I and Sma I, gel purified, and ligated at 1:3 vector/insert ratio. These manipulations resulted in the construction of (A) FabV-containing pUC19, (B) FabI-containing pUC19, (C) FabV-containing pSA-HP24, and (D) FabI-containing pBR322 plasmid vectors.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Purification

4) Product Images from "A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs"

Article Title: A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

Journal: mAbs

doi: 10.1080/19420862.2016.1277302

Cloning of circularly permutated Calmodulin variants. (1) Excision of gene encoding for calmodulin (CaM) with Bam HI from transfer vector pJ201-CaM. (2) Circularisation of linear gene with T4 DNA Ligase and amplification of permutated variants by PCR with appropriate oligonucleotide pairs. (3) Restriction of amplified CaM-variants with Nhe I and Eco RV and subsequent cloning into target vector (4) encoding for scFv. Abbreviations, P-rev: reverse-oligonucleotide with Eco RV-overhang; P-fwd: forward-oligonucleotide with Nhe I-overhang. Ptac: tac-promoter; PelB: signal peptide (PhoA was used in case of anti-CD20 scFv); V 1 / V 2 : heavy or light chain variable domain (V 1 = VH and V 2 = VL in anti-CD4, anti-CD14 and anti-biotin scFv; V 1 = VL and V 2 = VH in anti-CD20 and anti-FITC scFv); Nhe I/ Eco RV: restriction sites; 2xHis 6 : histidine-tag; rrnB T1: transcription terminator; Col E1 ori: origin of replication; laqIq: laqIq-promoter; kan: kanamycin resistance; CD: cluster of differentiation; FITC: fluorescein isothiocyanate.
Figure Legend Snippet: Cloning of circularly permutated Calmodulin variants. (1) Excision of gene encoding for calmodulin (CaM) with Bam HI from transfer vector pJ201-CaM. (2) Circularisation of linear gene with T4 DNA Ligase and amplification of permutated variants by PCR with appropriate oligonucleotide pairs. (3) Restriction of amplified CaM-variants with Nhe I and Eco RV and subsequent cloning into target vector (4) encoding for scFv. Abbreviations, P-rev: reverse-oligonucleotide with Eco RV-overhang; P-fwd: forward-oligonucleotide with Nhe I-overhang. Ptac: tac-promoter; PelB: signal peptide (PhoA was used in case of anti-CD20 scFv); V 1 / V 2 : heavy or light chain variable domain (V 1 = VH and V 2 = VL in anti-CD4, anti-CD14 and anti-biotin scFv; V 1 = VL and V 2 = VH in anti-CD20 and anti-FITC scFv); Nhe I/ Eco RV: restriction sites; 2xHis 6 : histidine-tag; rrnB T1: transcription terminator; Col E1 ori: origin of replication; laqIq: laqIq-promoter; kan: kanamycin resistance; CD: cluster of differentiation; FITC: fluorescein isothiocyanate.

Techniques Used: Clone Assay, Chick Chorioallantoic Membrane Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction

5) Product Images from "CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say"

Article Title: CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say

Journal: PLoS ONE

doi: 10.1371/journal.pone.0224857

Culex quinquefasciatus kmo gene and mutations observed. Structure of CPIJ017147 as annotated in Vectorbase, sequences of exon 2 through 5 were confirmed in our lab strain and three sgRNA targets in exon 5 were selected (A). White eyed G 1 individuals were collected for genomic DNA extraction, PCR, and sequencing of the kmo locus. All sequenced mutations are presented (B) followed by the size of the indel, and in brackets the number of sequenced alleles with that mutation. All sequenced alleles contained deletions derived from sgRNAs 935 and 936.
Figure Legend Snippet: Culex quinquefasciatus kmo gene and mutations observed. Structure of CPIJ017147 as annotated in Vectorbase, sequences of exon 2 through 5 were confirmed in our lab strain and three sgRNA targets in exon 5 were selected (A). White eyed G 1 individuals were collected for genomic DNA extraction, PCR, and sequencing of the kmo locus. All sequenced mutations are presented (B) followed by the size of the indel, and in brackets the number of sequenced alleles with that mutation. All sequenced alleles contained deletions derived from sgRNAs 935 and 936.

Techniques Used: DNA Extraction, Polymerase Chain Reaction, Sequencing, Mutagenesis, Derivative Assay

6) Product Images from "A Targeted Vaccine against COVID-19: S1-Fc Vaccine Targeting the Antigen-Presenting Cell Compartment Elicits Protection against SARS-CoV-2 Infection"

Article Title: A Targeted Vaccine against COVID-19: S1-Fc Vaccine Targeting the Antigen-Presenting Cell Compartment Elicits Protection against SARS-CoV-2 Infection

Journal: bioRxiv

doi: 10.1101/2020.06.29.178616

Production and secretion of S1-Fc-protein antigen by muscle cells. ( A ) Linear dsDNA encoding S1-Fc was generated by PCR-based amplification and subjected to analytical DNA gel electrophoresis, assessing accurate size and excluding undesired intermediate product. ( B ) Cellular internalization of fluorescently labeled dsDNA encoding S1-Fc was assessed by flow cytometry immediately after electroporation procedure, gating on dsDNA encoding S1-Fc + cells (red). ( C ) Cellular internalization of S1-Fc-dsDNA was validated by confocal microscopy. Scale, 50 μm. ( D ) Produced S1-Fc protein secreted by C2C12 muscle cells within 96 h after electroporation was assessed from collected cell supernatant and analyzed by MSD assay. SD shown. T-test: **) P
Figure Legend Snippet: Production and secretion of S1-Fc-protein antigen by muscle cells. ( A ) Linear dsDNA encoding S1-Fc was generated by PCR-based amplification and subjected to analytical DNA gel electrophoresis, assessing accurate size and excluding undesired intermediate product. ( B ) Cellular internalization of fluorescently labeled dsDNA encoding S1-Fc was assessed by flow cytometry immediately after electroporation procedure, gating on dsDNA encoding S1-Fc + cells (red). ( C ) Cellular internalization of S1-Fc-dsDNA was validated by confocal microscopy. Scale, 50 μm. ( D ) Produced S1-Fc protein secreted by C2C12 muscle cells within 96 h after electroporation was assessed from collected cell supernatant and analyzed by MSD assay. SD shown. T-test: **) P

Techniques Used: Generated, Polymerase Chain Reaction, Amplification, DNA Gel Electrophoresis, Labeling, Flow Cytometry, Electroporation, Confocal Microscopy, Produced

7) Product Images from "Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli"

Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-13-107

Construction and verification of pSA-Hp24-6His vector. We PCR amplified the p24 gene from pNL4.3 and cloned at Nde I/ Sac I restriction sites in inversely PCRed pMXB10 vector (A) . Agarose gel electrophoresis analysis (B) . Lane 1, 100 bp DNA ladder (NEB#N0467S); Lane 2, PCR amplified 714 bp p24; Lane 3, inversely PCRed 5.91 kb pSA vector; Lane 4, representative colony PCR (806 bp); Lane 5, restriction analysis of pSA-Hp24-6His with Nde I/ Sac I (5.89 kb vector backbone + 702 bp insert) ; Lane 6, 1 kb DNA ladder (NEB #N0468S).
Figure Legend Snippet: Construction and verification of pSA-Hp24-6His vector. We PCR amplified the p24 gene from pNL4.3 and cloned at Nde I/ Sac I restriction sites in inversely PCRed pMXB10 vector (A) . Agarose gel electrophoresis analysis (B) . Lane 1, 100 bp DNA ladder (NEB#N0467S); Lane 2, PCR amplified 714 bp p24; Lane 3, inversely PCRed 5.91 kb pSA vector; Lane 4, representative colony PCR (806 bp); Lane 5, restriction analysis of pSA-Hp24-6His with Nde I/ Sac I (5.89 kb vector backbone + 702 bp insert) ; Lane 6, 1 kb DNA ladder (NEB #N0468S).

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Agarose Gel Electrophoresis

8) Product Images from "Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks"

Article Title: Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2017.02.006

Generation of Different Donor Templates and px459-mRFP Target (A) One kilobase of double-stranded DNA (dsDNA) donor templates was generated with varying homology sequence overlaps on the 5′ and 3′ side of the mutation site. (B) Donor templates were generated as plasmid, linearized plasmid with 5′ or 3′ backbone overhang, or PCR product. (C) mRFP gene sequence was cloned into the px459 expression vector instead of the puromycin resistance gene.
Figure Legend Snippet: Generation of Different Donor Templates and px459-mRFP Target (A) One kilobase of double-stranded DNA (dsDNA) donor templates was generated with varying homology sequence overlaps on the 5′ and 3′ side of the mutation site. (B) Donor templates were generated as plasmid, linearized plasmid with 5′ or 3′ backbone overhang, or PCR product. (C) mRFP gene sequence was cloned into the px459 expression vector instead of the puromycin resistance gene.

Techniques Used: Generated, Sequencing, Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Expressing

9) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

Journal: Genes and Environment

doi: 10.1186/s41021-018-0112-5

One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.
Figure Legend Snippet: One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.

Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Countercurrent Chromatography

10) Product Images from "Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies"

Article Title: Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0137466

pUC19- lacZα assembly assay. ( A ) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the lacZα gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene ( bla ) and the origin of replication. The fragments shared 50-bp homology at both ends. ( B ) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates ( S3 Table ). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. ( C ) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.
Figure Legend Snippet: pUC19- lacZα assembly assay. ( A ) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the lacZα gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene ( bla ) and the origin of replication. The fragments shared 50-bp homology at both ends. ( B ) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates ( S3 Table ). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. ( C ) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Concentration Assay, Standard Deviation

11) Product Images from "In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes"

Article Title: In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

Journal: Plants

doi: 10.3390/plants6040052

Amplification of selected target gene fragments from DNA extracted from in situ dark- or light-adapted mature pin oak ( Q. palustris ) leaves. The figure shows a representative example of PCR amplification of the 1300 bp rbcL , 500 bp STS_8561_ palustris and 209 bp STS_8461_ palustris sequence tagged site segments from dark- (D) and light-adapted (L) leaves, using extraction method M1 and M2, on a 2% agarose gel containing 0.5 µg/mL ethidium bromide. The low molecular weight bands (at about 175 bp) in the lane showing the amplified rbcL segment (M1D and M1L) are non-specific amplification products. A 2 kb DNA ladder was used for fragment sizing. Arrows indicate the position of the 1400, 500 and 200 bp bands in the marker line. To establish whether there is a quantitative difference between the efficiency of the amplification of fragments, apparent bands were excised from the gel and the DNA purified and quantified from the excised fragments. Yields of the 1300 bp rbcL amplicon fragments were of 325 and 227.6 ng, 500 bp STS_8561_ palustris of 375 and 388 ng and 209 bp STS_8461_ palustris of 420 and 426 ng, for dark- and light-adapted samples, respectively. No bands were observed for any of the samples using as template the DNA isolated by method M2, suggesting no amplification of the target gene fragments. Abbreviations stand for: D = dark-adapted; L = light-adapted; M1 = method 1; M2 = method 2.
Figure Legend Snippet: Amplification of selected target gene fragments from DNA extracted from in situ dark- or light-adapted mature pin oak ( Q. palustris ) leaves. The figure shows a representative example of PCR amplification of the 1300 bp rbcL , 500 bp STS_8561_ palustris and 209 bp STS_8461_ palustris sequence tagged site segments from dark- (D) and light-adapted (L) leaves, using extraction method M1 and M2, on a 2% agarose gel containing 0.5 µg/mL ethidium bromide. The low molecular weight bands (at about 175 bp) in the lane showing the amplified rbcL segment (M1D and M1L) are non-specific amplification products. A 2 kb DNA ladder was used for fragment sizing. Arrows indicate the position of the 1400, 500 and 200 bp bands in the marker line. To establish whether there is a quantitative difference between the efficiency of the amplification of fragments, apparent bands were excised from the gel and the DNA purified and quantified from the excised fragments. Yields of the 1300 bp rbcL amplicon fragments were of 325 and 227.6 ng, 500 bp STS_8561_ palustris of 375 and 388 ng and 209 bp STS_8461_ palustris of 420 and 426 ng, for dark- and light-adapted samples, respectively. No bands were observed for any of the samples using as template the DNA isolated by method M2, suggesting no amplification of the target gene fragments. Abbreviations stand for: D = dark-adapted; L = light-adapted; M1 = method 1; M2 = method 2.

Techniques Used: Amplification, In Situ, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Marker, Purification, Isolation

Amplification of a partial segment of the putative gene encoding for the isoprene synthase (IspS) enzyme. PCR products were separated on a 2% agarose gel stained with 0.5 µg/mL ethidium bromide. The arrow indicates the position of the 165 bp fragment of the putative IspS gene. Lanes 2 and 3 were loaded with representative reactions containing DNA templates isolated from dark-adapted leaves, using M1. Lanes 4 and 5 were loaded with representative reactions containing DNA templates isolated from light-adapted leaves, using M1. The white arrow annotates the position of the 165 bp fragments. D = dark-adapted; L = light-adapted.
Figure Legend Snippet: Amplification of a partial segment of the putative gene encoding for the isoprene synthase (IspS) enzyme. PCR products were separated on a 2% agarose gel stained with 0.5 µg/mL ethidium bromide. The arrow indicates the position of the 165 bp fragment of the putative IspS gene. Lanes 2 and 3 were loaded with representative reactions containing DNA templates isolated from dark-adapted leaves, using M1. Lanes 4 and 5 were loaded with representative reactions containing DNA templates isolated from light-adapted leaves, using M1. The white arrow annotates the position of the 165 bp fragments. D = dark-adapted; L = light-adapted.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Isolation

12) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

Journal: Genes and Environment

doi: 10.1186/s41021-018-0112-5

One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.
Figure Legend Snippet: One-pot synthesis of DNA repair substrate. a Experimental procedure for purification of pBS2/A:C omitting a column purification step. b Aliquots from various steps of the purification were subjected to 0.8% agarose gel electrophoresis, and the DNA substrates were visualized by staining with EtBr. Lane 1, pBS2-SDL; lane 2, Nt.BbvCI-treatment; lane 3, T4 DNA ligase-treatment; lane 4, Eco NI-treatment; lane 5, T5 exonuclease-treatment: lane 6, purified pBS2A:C by PCR purification kit. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. And the irreversibly denatured form was observed as the minor band shorter than the CCC band.

Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Countercurrent Chromatography

13) Product Images from "Bamboo tea: reduction of taxonomic complexity and application of DNA diagnostics based on rbcL and matK sequence data"

Article Title: Bamboo tea: reduction of taxonomic complexity and application of DNA diagnostics based on rbcL and matK sequence data

Journal: PeerJ

doi: 10.7717/peerj.2781

PCR diagnostics. Comparison of multiplex PCR results using rbcLa universal primers and diagnostic (ARMS) primer. On the left are the results using DNA templates derived from products P1 to P8 with primer specific for bamboo (B), lemongrass (L) and Dianthus (D). Next, fragment pattern predictions (PFP) for presence (+) and absence (−) of corresponding components are depicted. The rbcLa fragment with a size of around 600 bp represents the positive reaction control. A smaller fragment is the diagnostic fragment and indicates the presence of a particular component (e.g., 306 bp fragment for lemongrass). On the right are representative results using DNA templates derived from bamboo (B), lemongrass (L) and Dianthus (D) specimens. For the approximation of fragment size a 100 bp (NEB) size standard (M) was used.
Figure Legend Snippet: PCR diagnostics. Comparison of multiplex PCR results using rbcLa universal primers and diagnostic (ARMS) primer. On the left are the results using DNA templates derived from products P1 to P8 with primer specific for bamboo (B), lemongrass (L) and Dianthus (D). Next, fragment pattern predictions (PFP) for presence (+) and absence (−) of corresponding components are depicted. The rbcLa fragment with a size of around 600 bp represents the positive reaction control. A smaller fragment is the diagnostic fragment and indicates the presence of a particular component (e.g., 306 bp fragment for lemongrass). On the right are representative results using DNA templates derived from bamboo (B), lemongrass (L) and Dianthus (D) specimens. For the approximation of fragment size a 100 bp (NEB) size standard (M) was used.

Techniques Used: Polymerase Chain Reaction, Multiplex Assay, Diagnostic Assay, Derivative Assay

14) Product Images from "Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System"

Article Title: Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System

Journal: Bio-protocol

doi: 10.21769/BioProtoc.2431

Illustration for PCR validation. Forward and reverse primers anneal upstream and downstream to geneX (green). PCR amplification on the edited phage results in a smaller DNA product. PCR amplification of the WT phage or phage with deletion of a fragment encoding the corresponding protospacer results in a full length DNA product. Black square represents gel electrophoresis, white bands represent DNA products.
Figure Legend Snippet: Illustration for PCR validation. Forward and reverse primers anneal upstream and downstream to geneX (green). PCR amplification on the edited phage results in a smaller DNA product. PCR amplification of the WT phage or phage with deletion of a fragment encoding the corresponding protospacer results in a full length DNA product. Black square represents gel electrophoresis, white bands represent DNA products.

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

15) Product Images from "Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita"

Article Title: Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita

Journal: Scientific Reports

doi: 10.1038/s41598-018-37321-z

Relative transcript levels of QQ-ORFs in A . aurita polyps and ephyrae. Relative transcript levels of aaqq1 , aaqq2 and aaqq3 were determined by qRT-PCR analysis in A . aurita polyps and ephyrae with at least three independent biological experiments, each with three technical replicates. ( A ) Polyps and ephyrae were kept under native and germ-free conditions. ( B–E ) Polyps and ephyrae were kept under native ( B+D ) and germ-free ( C+E ) conditions, additionally challenged with selected bacteria, K . oxytoca , V . parahaemolyticus and P . tunicata . Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene actin .
Figure Legend Snippet: Relative transcript levels of QQ-ORFs in A . aurita polyps and ephyrae. Relative transcript levels of aaqq1 , aaqq2 and aaqq3 were determined by qRT-PCR analysis in A . aurita polyps and ephyrae with at least three independent biological experiments, each with three technical replicates. ( A ) Polyps and ephyrae were kept under native and germ-free conditions. ( B–E ) Polyps and ephyrae were kept under native ( B+D ) and germ-free ( C+E ) conditions, additionally challenged with selected bacteria, K . oxytoca , V . parahaemolyticus and P . tunicata . Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene actin .

Techniques Used: Quantitative RT-PCR

Characterization of A . aurita QQ ORFs. ( A ) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1 , aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). ( B ) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. ( C ) were used for functional protein analysis. ( D ) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.
Figure Legend Snippet: Characterization of A . aurita QQ ORFs. ( A ) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1 , aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). ( B ) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. ( C ) were used for functional protein analysis. ( D ) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.

Techniques Used: Clone Assay, Purification, Affinity Chromatography, SDS Page, Functional Assay, Polymerase Chain Reaction, Amplification

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Amplification:

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In Vitro:

Article Title: CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say
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Purification:

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Real-time Polymerase Chain Reaction:

Article Title: Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita
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Polymerase Chain Reaction:

Article Title: Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita
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Article Title: A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs
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Plasmid Preparation:

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    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.
    Pcr Clean Up Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL nucleospin gdna clean up xs kit
    Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) <t>NucleoSpin</t> ® <t>gDNA</t> Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.
    Nucleospin Gdna Clean Up Xs Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    Recovery of three distinct coat-colour mutations by CRISPR/Cas. ( a ) Schematic illustration of three coat-colour mutations in rats. albino ( Tyr c ): SNP missense mutation in the Tyr gene. non-agouti ( Asip a ): 19-bp deletion in exon 2 of the Agouti signalling protein ( Asip ) gene. hooded ( Kit h ): integration of an 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. ( b ) Coat-colour phenotypes ( C, a, h ) recovered from albino by injecting gRNA: Tyr c , Cas9 mRNA, and ssODN of the Tyr C allele into F344 rat embryos ( c, a, h ). ( c ) Sequence analysis of the targeted Tyr exon 2 in the injected F344 rats. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise HDR-mediated SNP exchange of Tyr C . ( d ) Recovery of the non-agouti phenotype by injecting gRNA: Asip a , Cas9 mRNA, and ssODN of the Asip A allele into F344 rat embryos. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise short DNA fragment integration of the Asip A gene . ( e ) Recovery of the hooded mutation by injecting gRNA: Kit h -1 , gRNA: Kit h -2 , and ssODN of the Kit H allele into F344 rat embryos. Cas9 and two gRNAs with ssODN mediated the introduction of indel mutations at the targeted Kit h -1 locus, and the precise large deletion between the two cutting edges of Kit H . ( f ) PCR analysis of the injected F344 rats using primers designed against each outer side of the two LTR sequences. M: DNA marker phiX174-HaeIII digest.

    Journal: Nature Communications

    Article Title: Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform

    doi: 10.1038/ncomms5240

    Figure Lengend Snippet: Recovery of three distinct coat-colour mutations by CRISPR/Cas. ( a ) Schematic illustration of three coat-colour mutations in rats. albino ( Tyr c ): SNP missense mutation in the Tyr gene. non-agouti ( Asip a ): 19-bp deletion in exon 2 of the Agouti signalling protein ( Asip ) gene. hooded ( Kit h ): integration of an 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. ( b ) Coat-colour phenotypes ( C, a, h ) recovered from albino by injecting gRNA: Tyr c , Cas9 mRNA, and ssODN of the Tyr C allele into F344 rat embryos ( c, a, h ). ( c ) Sequence analysis of the targeted Tyr exon 2 in the injected F344 rats. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise HDR-mediated SNP exchange of Tyr C . ( d ) Recovery of the non-agouti phenotype by injecting gRNA: Asip a , Cas9 mRNA, and ssODN of the Asip A allele into F344 rat embryos. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise short DNA fragment integration of the Asip A gene . ( e ) Recovery of the hooded mutation by injecting gRNA: Kit h -1 , gRNA: Kit h -2 , and ssODN of the Kit H allele into F344 rat embryos. Cas9 and two gRNAs with ssODN mediated the introduction of indel mutations at the targeted Kit h -1 locus, and the precise large deletion between the two cutting edges of Kit H . ( f ) PCR analysis of the injected F344 rats using primers designed against each outer side of the two LTR sequences. M: DNA marker phiX174-HaeIII digest.

    Article Snippet: To prepare Cas9 mRNA and gRNA, T7-promoter Cas9 and gRNA expression plasmids were linearized with Xho I and Hind III, respectively, and extracted with NucleoSpin Gel and PCR Clean-up kits (Macherey-Nagel, Düren, Germany).

    Techniques: CRISPR, Mutagenesis, Sequencing, Injection, Polymerase Chain Reaction, Marker

    Relative transcript levels of QQ-ORFs in A . aurita polyps and ephyrae. Relative transcript levels of aaqq1 , aaqq2 and aaqq3 were determined by qRT-PCR analysis in A . aurita polyps and ephyrae with at least three independent biological experiments, each with three technical replicates. ( A ) Polyps and ephyrae were kept under native and germ-free conditions. ( B–E ) Polyps and ephyrae were kept under native ( B+D ) and germ-free ( C+E ) conditions, additionally challenged with selected bacteria, K . oxytoca , V . parahaemolyticus and P . tunicata . Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene actin .

    Journal: Scientific Reports

    Article Title: Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita

    doi: 10.1038/s41598-018-37321-z

    Figure Lengend Snippet: Relative transcript levels of QQ-ORFs in A . aurita polyps and ephyrae. Relative transcript levels of aaqq1 , aaqq2 and aaqq3 were determined by qRT-PCR analysis in A . aurita polyps and ephyrae with at least three independent biological experiments, each with three technical replicates. ( A ) Polyps and ephyrae were kept under native and germ-free conditions. ( B–E ) Polyps and ephyrae were kept under native ( B+D ) and germ-free ( C+E ) conditions, additionally challenged with selected bacteria, K . oxytoca , V . parahaemolyticus and P . tunicata . Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene actin .

    Article Snippet: Before use of cDNA in quantitative PCR, samples were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). qRT-PCRs were performed with three independent RNA preparations each with three technical replicates on an Applied Biosystems 7300 Real-Time PCR System using cDNA prepared with PlatinumR SYBRR Green qPCR SuperMix-UDG with ROX (Thermo Fisher Scientific, Darmstadt, Germany).

    Techniques: Quantitative RT-PCR

    Characterization of A . aurita QQ ORFs. ( A ) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1 , aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). ( B ) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. ( C ) QQ proteins were analyzed using secondary structure prediction software PredictProtein ( http://www.predictprotein.org/ ) with following visual output in Profsec: yellow, strand; red, helix. InterProScan ( http://www.ebi.ac.uk/Tools/pfa/iprscan/ ) and GlobPlot ( http://globplot.embl.de/ ) were used for functional protein analysis. ( D ) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.

    Journal: Scientific Reports

    Article Title: Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita

    doi: 10.1038/s41598-018-37321-z

    Figure Lengend Snippet: Characterization of A . aurita QQ ORFs. ( A ) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1 , aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). ( B ) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. ( C ) QQ proteins were analyzed using secondary structure prediction software PredictProtein ( http://www.predictprotein.org/ ) with following visual output in Profsec: yellow, strand; red, helix. InterProScan ( http://www.ebi.ac.uk/Tools/pfa/iprscan/ ) and GlobPlot ( http://globplot.embl.de/ ) were used for functional protein analysis. ( D ) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.

    Article Snippet: Before use of cDNA in quantitative PCR, samples were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). qRT-PCRs were performed with three independent RNA preparations each with three technical replicates on an Applied Biosystems 7300 Real-Time PCR System using cDNA prepared with PlatinumR SYBRR Green qPCR SuperMix-UDG with ROX (Thermo Fisher Scientific, Darmstadt, Germany).

    Techniques: Clone Assay, Purification, Affinity Chromatography, SDS Page, Software, Functional Assay, Polymerase Chain Reaction, Amplification

    Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) NucleoSpin ® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.

    Journal: PLoS ONE

    Article Title: Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

    doi: 10.1371/journal.pone.0158698

    Figure Lengend Snippet: Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) NucleoSpin ® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.

    Article Snippet: Finally, the NucleoSpin® gDNA Clean-up XS Kit provided greater efficiency of BRAF amplification but did not completely resolve the difficulties presented by these types of samples, notably when higher concentrations of melanin were added ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Cell Culture, Electrophoresis, Staining, Molecular Weight