dna clean kit  (Zymo Research)


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    Name:
    Select a Size DNA Clean Concentrator Kit
    Description:
    The Select a Size DNA Clean Concentrator Kit Select a Size DCC provides the quickest and easiest method for purifying a desired range of DNA fragments sizes from library preps PCR endonuclease digestions ligations etc Simply adjust the binding conditions for the desired cutoff bind wash and elute Selectively recover 300 bp 200 bp 150 bp 100 bp 50 bp DNA fragments or perform a double size selection Unique Zymo Spin column technology yields high quality DNA in just minutes that is suitable for next generation sequencing PCR and other downstream applications The entire purification procedure can be performed in as little as 7 minutes for 2 preps or 20 minutes for 24 samples See figures below
    Catalog Number:
    d4080
    Price:
    None
    Applications:
    DNA Clean-up & Size Selection
    Size:
    25 units
    Category:
    Life Science Reagents and Media
    Buy from Supplier


    Structured Review

    Zymo Research dna clean kit
    Select a Size DNA Clean Concentrator Kit
    The Select a Size DNA Clean Concentrator Kit Select a Size DCC provides the quickest and easiest method for purifying a desired range of DNA fragments sizes from library preps PCR endonuclease digestions ligations etc Simply adjust the binding conditions for the desired cutoff bind wash and elute Selectively recover 300 bp 200 bp 150 bp 100 bp 50 bp DNA fragments or perform a double size selection Unique Zymo Spin column technology yields high quality DNA in just minutes that is suitable for next generation sequencing PCR and other downstream applications The entire purification procedure can be performed in as little as 7 minutes for 2 preps or 20 minutes for 24 samples See figures below
    https://www.bioz.com/result/dna clean kit/product/Zymo Research
    Average 99 stars, based on 622 article reviews
    Price from $9.99 to $1999.99
    dna clean kit - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification"

    Article Title: Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification

    Journal: Malaria Journal

    doi: 10.1186/s12936-020-03259-9

    Success rate of molecular analysis using DNA extracted from RDTs
    Figure Legend Snippet: Success rate of molecular analysis using DNA extracted from RDTs

    Techniques Used:

    Utility of DNA from RDTs for molecular analysis
    Figure Legend Snippet: Utility of DNA from RDTs for molecular analysis

    Techniques Used:

    2) Product Images from "Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer"

    Article Title: Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-8-128

    Effect of DAC concentration on DNA methylation and reactivation of p57 and p16 tumor suppressor genes . A), HL-60 cells were treated with the indicated concentrations of DAC for 48 h. Total DNA was isolated at 72 h and the LINE-1 element gene methylation status was analyzed by COBRA assay. B), HL-60 cells were treated with the indicated concentrations of DAC for 48 h and total RNA was isolated at 72 h. Gene expression of 18 S ribosomal RNA gene and p57 were analyzed by RT-PCR. The amount of DNA amplified during the exponential phase of PCR was analyzed by quantification of amplified DNA by an Agilent 2100 Bioanalyzer. The control cells are HL-60 with no drug treatment. Control vs DAC 100 ng/ml or 1,000 ng/ml p
    Figure Legend Snippet: Effect of DAC concentration on DNA methylation and reactivation of p57 and p16 tumor suppressor genes . A), HL-60 cells were treated with the indicated concentrations of DAC for 48 h. Total DNA was isolated at 72 h and the LINE-1 element gene methylation status was analyzed by COBRA assay. B), HL-60 cells were treated with the indicated concentrations of DAC for 48 h and total RNA was isolated at 72 h. Gene expression of 18 S ribosomal RNA gene and p57 were analyzed by RT-PCR. The amount of DNA amplified during the exponential phase of PCR was analyzed by quantification of amplified DNA by an Agilent 2100 Bioanalyzer. The control cells are HL-60 with no drug treatment. Control vs DAC 100 ng/ml or 1,000 ng/ml p

    Techniques Used: Concentration Assay, DNA Methylation Assay, Isolation, Methylation, Combined Bisulfite Restriction Analysis Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    3) Product Images from "Large diverse population cohorts of hiPSCs and derived hepatocyte-like cells reveal functional genetic variation at blood lipid-associated loci"

    Article Title: Large diverse population cohorts of hiPSCs and derived hepatocyte-like cells reveal functional genetic variation at blood lipid-associated loci

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2017.03.017

    Evidence for rs2277862- CPNE1 as a Functional SNP-Gene Set (A) Schematics of human chromosome 20q11 locus showing the relative positions of rs2277862, CPNE1 , and ERGIC3 and mouse chromosome 2qH1 locus showing the relative positions of rs27324996, Cpne1 , and Ergic3 . (B) Top panels: heterozygous knock-in of rs2277862 minor allele with a single-strand DNA oligonucleotide. Representative indels in non-knock-in clones are also shown. Bottom panels: homozygous 38-bp deletions (“knockout” or Δ38/Δ38) encompassing rs2277862 using a dual gRNA approach. A representative agarose gel of PCR amplicons is shown. The protospacers are underlined, the PAMs are bolded, and the SNP position is indicated in red. (C) Left panels: gene expression in undifferentiated HUES 8 cells (n = 2 wild-type clones and 1 knock-in clone; 6 wells per clone) and differentiated HUES 8 HLCs (n = 2 wild-type clones and 1 knock-in clone; 6 wells per clone) normalized to mean expression level in wild-type clones. Right panels: gene expression in undifferentiated HUES 8 cells (n = 10 wild-type and 10 knockout clones, 3 wells per clone) and undifferentiated H7 cells (n = 8 wild-type and 6 knockout clones, 3 wells per clone) normalized to mean expression level in wild-type clones. (D) CRISPR interference at the rs2277862 site. The three gRNA protospacers are underlined, the PAMs are bolded, and the SNP position is indicated in red. The graphs show gene expression, normalized to mean expression level in control cells, in HEK 293T cells transfected with catalytically dead Cas9 (dCas9) with the gRNAs (either singly or in combination, 3 wells per condition). Control cells were transfected with dCas9 without an accompanying gRNA. (E) The noncoding rs2277862 site is well conserved in mouse, including allelic variants of the SNP itself, with the murine equivalent being rs27324996. The SNP position is indicated in red, non-conserved nucleotides are indicated in blue, the gRNA protospacer used to generate the knock-in mouse is underlined, and the PAM is bolded. The electropherogram is from a mouse in which the minor allele of rs2277862/rs27324996 (T) has been knocked into one chromosome, along with four non-conserved nucleotides to “humanize” the site. (F) Gene expression in liver from littermates of the C57BL/6J background (n = 18 wild-type mice and 10 homozygous knock-in mice), normalized to mean expression level in wild-type mice. Data are displayed as means and s.e.m. P -values were calculated with two-tailed Welch’s t -tests.
    Figure Legend Snippet: Evidence for rs2277862- CPNE1 as a Functional SNP-Gene Set (A) Schematics of human chromosome 20q11 locus showing the relative positions of rs2277862, CPNE1 , and ERGIC3 and mouse chromosome 2qH1 locus showing the relative positions of rs27324996, Cpne1 , and Ergic3 . (B) Top panels: heterozygous knock-in of rs2277862 minor allele with a single-strand DNA oligonucleotide. Representative indels in non-knock-in clones are also shown. Bottom panels: homozygous 38-bp deletions (“knockout” or Δ38/Δ38) encompassing rs2277862 using a dual gRNA approach. A representative agarose gel of PCR amplicons is shown. The protospacers are underlined, the PAMs are bolded, and the SNP position is indicated in red. (C) Left panels: gene expression in undifferentiated HUES 8 cells (n = 2 wild-type clones and 1 knock-in clone; 6 wells per clone) and differentiated HUES 8 HLCs (n = 2 wild-type clones and 1 knock-in clone; 6 wells per clone) normalized to mean expression level in wild-type clones. Right panels: gene expression in undifferentiated HUES 8 cells (n = 10 wild-type and 10 knockout clones, 3 wells per clone) and undifferentiated H7 cells (n = 8 wild-type and 6 knockout clones, 3 wells per clone) normalized to mean expression level in wild-type clones. (D) CRISPR interference at the rs2277862 site. The three gRNA protospacers are underlined, the PAMs are bolded, and the SNP position is indicated in red. The graphs show gene expression, normalized to mean expression level in control cells, in HEK 293T cells transfected with catalytically dead Cas9 (dCas9) with the gRNAs (either singly or in combination, 3 wells per condition). Control cells were transfected with dCas9 without an accompanying gRNA. (E) The noncoding rs2277862 site is well conserved in mouse, including allelic variants of the SNP itself, with the murine equivalent being rs27324996. The SNP position is indicated in red, non-conserved nucleotides are indicated in blue, the gRNA protospacer used to generate the knock-in mouse is underlined, and the PAM is bolded. The electropherogram is from a mouse in which the minor allele of rs2277862/rs27324996 (T) has been knocked into one chromosome, along with four non-conserved nucleotides to “humanize” the site. (F) Gene expression in liver from littermates of the C57BL/6J background (n = 18 wild-type mice and 10 homozygous knock-in mice), normalized to mean expression level in wild-type mice. Data are displayed as means and s.e.m. P -values were calculated with two-tailed Welch’s t -tests.

    Techniques Used: Functional Assay, Knock-In, Clone Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Knock-Out, CRISPR, Transfection, Mouse Assay, Two Tailed Test

    4) Product Images from "RRHP: a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution"

    Article Title: RRHP: a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution

    Journal: Genome Biology

    doi: 10.1186/s13059-014-0456-5

    Workflow of the RRHP assay. Genomic DNA was first digested by restriction endonuclease MspI and then ligated with the modified adapters such that the MspI site is reconstituted at the junction of P5 adapter and fragment but not at the junction of P7 and fragment. The adapter-ligated fragments are either homo- or hetero-adapterized. Homo-adapterized fragments have the same adapters at both ends (P5-P5 or P7-P7), which will form an inhibitory stem-loop hairpin preventing itself from further amplification, whereas the hetero-adapterized fragments have different adapters (P5-P7). All ligated library fragments are glucosylated with β-glucosyltransferase (β-GT). Fragments presenting 5hmC at the junction and, thus glucosylated, are resistant to a second MspI digestion while fragments with unmodified C or 5mC are cleaved, removing the P5 adapter. Only the fragments with both P5 and P7 adapters after the second MspI digestion will be amplified and sequenced.
    Figure Legend Snippet: Workflow of the RRHP assay. Genomic DNA was first digested by restriction endonuclease MspI and then ligated with the modified adapters such that the MspI site is reconstituted at the junction of P5 adapter and fragment but not at the junction of P7 and fragment. The adapter-ligated fragments are either homo- or hetero-adapterized. Homo-adapterized fragments have the same adapters at both ends (P5-P5 or P7-P7), which will form an inhibitory stem-loop hairpin preventing itself from further amplification, whereas the hetero-adapterized fragments have different adapters (P5-P7). All ligated library fragments are glucosylated with β-glucosyltransferase (β-GT). Fragments presenting 5hmC at the junction and, thus glucosylated, are resistant to a second MspI digestion while fragments with unmodified C or 5mC are cleaved, removing the P5 adapter. Only the fragments with both P5 and P7 adapters after the second MspI digestion will be amplified and sequenced.

    Techniques Used: Modification, Amplification

    5) Product Images from "Bacillus anthracis Virulence Regulator AtxA Binds Specifically to the pagA Promoter Region"

    Article Title: Bacillus anthracis Virulence Regulator AtxA Binds Specifically to the pagA Promoter Region

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00569-19

    EMSA analysis indicates that the predicted SLII is important for DNA binding. (A) Nucleotide sequences of the pRSP pagA promoter region. The sequence shown is located immediately upstream of the translation start codon ATG (uppercase bold). The −35 and −10 regions of a σ A ). The hypothesized SLI is presented in bold and SLII is in bold italic. Transcription start site A is indicated in uppercase bold italic. (B) Map of PAG4-1 and PAG4-2 fragments within PAG4 in relation to the hypothesized stem-loops. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (C) EMSA on a 2% agarose EABE gel with AtxA WT incubated with PAG4 fragment and unlabeled competitor of either PAG4-1 or PAG4-2 generated via PCR. An excess of 10×, 30×, 60×, and 100× was added in increasing amounts to the EMSAs. (D) Map of RPAGΔSLI and RPAGΔSLII fragments in relation to the hypothesized stem-loops on the labeled RPAG700 fragment. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (E) EMSA on a 2% agarose EABE gel with AtxA WT incubated with the RPAG700 fragment and unlabeled competitor of either RPAGΔSLI or RPAGΔSLII generated via PCR. Excesses of 10×, 30×, and 60× were added in increasing amounts to the EMSA mixtures.
    Figure Legend Snippet: EMSA analysis indicates that the predicted SLII is important for DNA binding. (A) Nucleotide sequences of the pRSP pagA promoter region. The sequence shown is located immediately upstream of the translation start codon ATG (uppercase bold). The −35 and −10 regions of a σ A ). The hypothesized SLI is presented in bold and SLII is in bold italic. Transcription start site A is indicated in uppercase bold italic. (B) Map of PAG4-1 and PAG4-2 fragments within PAG4 in relation to the hypothesized stem-loops. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (C) EMSA on a 2% agarose EABE gel with AtxA WT incubated with PAG4 fragment and unlabeled competitor of either PAG4-1 or PAG4-2 generated via PCR. An excess of 10×, 30×, 60×, and 100× was added in increasing amounts to the EMSAs. (D) Map of RPAGΔSLI and RPAGΔSLII fragments in relation to the hypothesized stem-loops on the labeled RPAG700 fragment. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (E) EMSA on a 2% agarose EABE gel with AtxA WT incubated with the RPAG700 fragment and unlabeled competitor of either RPAGΔSLI or RPAGΔSLII generated via PCR. Excesses of 10×, 30×, and 60× were added in increasing amounts to the EMSA mixtures.

    Techniques Used: Binding Assay, Sequencing, Incubation, Generated, Polymerase Chain Reaction, Labeling

    6) Product Images from "The Clinical Influence of Autophagy-Associated Proteins on Human Lung Cancer"

    Article Title: The Clinical Influence of Autophagy-Associated Proteins on Human Lung Cancer

    Journal: Disease Markers

    doi: 10.1155/2018/8314963

    (a) Methylation status of LC3A in 5 lung cancer cell lines and HBEC. Bisulfite sequencing (BS) showed a heterogeneous methylation pattern in the promoter region and exon 4 of the LC3A gene in lung cancer cell lines. White: unmethylated; grey: partially methylated or only one allele is methylated; black: totally methylated or two alleles are methylated. 1–6: 6 CpG sites in promoter region; 1–8: 8 CpG sites in exon 4. (b) Representative results from methylation-specific PCR (MSP) showing that LC3A was unmethylated in cases 1, 4, and 5 while partially methylated in cases 3, 6, 7, and 8. Case 2 was excluded for the statistical analysis (see Supplementary Table 3 ), since no PCR products were observed most probably due to degraded genomic DNA. U: unmethylated; M: methylated.
    Figure Legend Snippet: (a) Methylation status of LC3A in 5 lung cancer cell lines and HBEC. Bisulfite sequencing (BS) showed a heterogeneous methylation pattern in the promoter region and exon 4 of the LC3A gene in lung cancer cell lines. White: unmethylated; grey: partially methylated or only one allele is methylated; black: totally methylated or two alleles are methylated. 1–6: 6 CpG sites in promoter region; 1–8: 8 CpG sites in exon 4. (b) Representative results from methylation-specific PCR (MSP) showing that LC3A was unmethylated in cases 1, 4, and 5 while partially methylated in cases 3, 6, 7, and 8. Case 2 was excluded for the statistical analysis (see Supplementary Table 3 ), since no PCR products were observed most probably due to degraded genomic DNA. U: unmethylated; M: methylated.

    Techniques Used: Methylation, Methylation Sequencing, Polymerase Chain Reaction

    7) Product Images from "Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria"

    Article Title: Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

    Journal: mSystems

    doi: 10.1128/mSystems.00143-17

    Golden Gate assembly of transposon delivery vectors from part vectors. (A to E) The five part vectors that are used for Golden Gate assembly of the magic pool transposon delivery vectors: part1 vector, part2 vector, part3 vector, barcoded part4 vector, and part5 vector (not drawn to scale). We show the sequences of the 4-nucleotide overhangs for Golden Gate assembly. (F) The transposon delivery vector with DNA barcode. ColE1 is the replication origin ColE1. cat is the chloramphenicol resistance gene. GFP is green fluorescent protein. oriT is the origin of transfer. AmpR is the beta-lactam resistance gene. R6K is a conditional replication origin. N20 is random 20-nucleotide DNA barcode.
    Figure Legend Snippet: Golden Gate assembly of transposon delivery vectors from part vectors. (A to E) The five part vectors that are used for Golden Gate assembly of the magic pool transposon delivery vectors: part1 vector, part2 vector, part3 vector, barcoded part4 vector, and part5 vector (not drawn to scale). We show the sequences of the 4-nucleotide overhangs for Golden Gate assembly. (F) The transposon delivery vector with DNA barcode. ColE1 is the replication origin ColE1. cat is the chloramphenicol resistance gene. GFP is green fluorescent protein. oriT is the origin of transfer. AmpR is the beta-lactam resistance gene. R6K is a conditional replication origin. N20 is random 20-nucleotide DNA barcode.

    Techniques Used: Plasmid Preparation

    Overview of the magic pool strategy. (A) Basic structure of a typical transposon delivery vector (not drawn to scale). The inverted repeat (IR) for the specific transposase is indicated. We dissected the transposon delivery vector into five different parts compatible with Golden Gate assembly, and the different parts are indicated by different colors. (B) General workflow of construction and application of magic pools. In step 1, variants of the five different parts are designed, cloned into a part-holding vector, confirmed by sequencing, and archived. In step 2, the part vectors are mixed and assembled using Golden Gate assembly to produce the magic pools of transposon delivery vectors. In step 3, the magic pool vectors are characterized by DNA sequencing whereby each unique DNA barcode (random 20-nucleotide DNA barcode [N20]) is linked to a specific combination of parts. In step 4, preliminary mutant libraries of approximately 5,000 CFU are made using the magic pool, and TnSeq is performed to link the DNA barcode to the insertion site, thereby simultaneously assessing the efficacy of the vectors in the magic pool. ID, identification. In step 5, an effective vector is reassembled using the archived parts, fully barcoded with millions of random DNA barcodes, and a full RB-TnSeq transposon mutant library is constructed. oriT is the origin of transfer. AmpR is the beta-lactam resistance cassette. R6K is the conditional replication origin.
    Figure Legend Snippet: Overview of the magic pool strategy. (A) Basic structure of a typical transposon delivery vector (not drawn to scale). The inverted repeat (IR) for the specific transposase is indicated. We dissected the transposon delivery vector into five different parts compatible with Golden Gate assembly, and the different parts are indicated by different colors. (B) General workflow of construction and application of magic pools. In step 1, variants of the five different parts are designed, cloned into a part-holding vector, confirmed by sequencing, and archived. In step 2, the part vectors are mixed and assembled using Golden Gate assembly to produce the magic pools of transposon delivery vectors. In step 3, the magic pool vectors are characterized by DNA sequencing whereby each unique DNA barcode (random 20-nucleotide DNA barcode [N20]) is linked to a specific combination of parts. In step 4, preliminary mutant libraries of approximately 5,000 CFU are made using the magic pool, and TnSeq is performed to link the DNA barcode to the insertion site, thereby simultaneously assessing the efficacy of the vectors in the magic pool. ID, identification. In step 5, an effective vector is reassembled using the archived parts, fully barcoded with millions of random DNA barcodes, and a full RB-TnSeq transposon mutant library is constructed. oriT is the origin of transfer. AmpR is the beta-lactam resistance cassette. R6K is the conditional replication origin.

    Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, DNA Sequencing, Mutagenesis, Construct

    8) Product Images from "Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction"

    Article Title: Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.8

    PCR amplification of various sequences by synthetic Dpo4-5m. ( a ) PCR amplification of sequences with lengths ranging from 110 bp to 1 kb by synthetic Dpo4-5m for 35 cycles. The expected amplicon lengths are indicated above the lanes. Primer dimer bands can be observed below the main product bands. The extension time was set to 2 min per cycle for 110–300 bp sequences, 5 min for 400–600 bp sequences and 10 min for 700–1000 bp sequences. W/o template: negative control with primers and enzyme but without template; NC (110 bp)/NC (1 kb): negative control with 110 bp or 1 kb templates and primers but without enzyme. ( b ) PCR amplification of the 1.1 kb dpo4 gene by synthetic Dpo4-5 m for 35 cycles, with the extension time set to 10 min per cycle. ( c ) PCR amplification of the 120 bp 5S E. coli rRNA gene rrfB by synthetic Dpo4-5m for 35 cycles, with the extension time set to 2 min per cycle. ( d ) PCR amplification of the 1.5 kb 16S E. coli rRNA gene rrsC by synthetic Dpo4-5m for 35 cycles, with the extension time set to 15 min per cycle. A primer dimer band can be observed below the main product band. All the PCR products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. The expected amplicon lengths are indicated above the lanes. NC, negative control with template and primers but without enzyme. M, DNA marker.
    Figure Legend Snippet: PCR amplification of various sequences by synthetic Dpo4-5m. ( a ) PCR amplification of sequences with lengths ranging from 110 bp to 1 kb by synthetic Dpo4-5m for 35 cycles. The expected amplicon lengths are indicated above the lanes. Primer dimer bands can be observed below the main product bands. The extension time was set to 2 min per cycle for 110–300 bp sequences, 5 min for 400–600 bp sequences and 10 min for 700–1000 bp sequences. W/o template: negative control with primers and enzyme but without template; NC (110 bp)/NC (1 kb): negative control with 110 bp or 1 kb templates and primers but without enzyme. ( b ) PCR amplification of the 1.1 kb dpo4 gene by synthetic Dpo4-5 m for 35 cycles, with the extension time set to 10 min per cycle. ( c ) PCR amplification of the 120 bp 5S E. coli rRNA gene rrfB by synthetic Dpo4-5m for 35 cycles, with the extension time set to 2 min per cycle. ( d ) PCR amplification of the 1.5 kb 16S E. coli rRNA gene rrsC by synthetic Dpo4-5m for 35 cycles, with the extension time set to 15 min per cycle. A primer dimer band can be observed below the main product band. All the PCR products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. The expected amplicon lengths are indicated above the lanes. NC, negative control with template and primers but without enzyme. M, DNA marker.

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control, Agarose Gel Electrophoresis, Staining, Marker

    Assembly PCR by synthetic Dpo4-5m. ( a , b ) Assembly PCR using two long primers ( tC19Z -F115 and tC19Z -R113) with a 30 bp sequence overlap for 20 cycles to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. Exo I: treated by exonuclease I (which digests single-stranded DNA (ssDNA) but not dsDNA). ( c , d ) Three-step assembly PCR using six short primers ranging in lengths from 47 to 59 nt to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. The PCR cycle numbers for steps 1, 2 and 3 were 5, 10 and 20, respectively. The expected amplicon lengths of each step (88, 162 and 198 bp, respectively) are indicated above the lanes. Primer dimer bands can be observed below the main product bands. M, DNA marker.
    Figure Legend Snippet: Assembly PCR by synthetic Dpo4-5m. ( a , b ) Assembly PCR using two long primers ( tC19Z -F115 and tC19Z -R113) with a 30 bp sequence overlap for 20 cycles to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. Exo I: treated by exonuclease I (which digests single-stranded DNA (ssDNA) but not dsDNA). ( c , d ) Three-step assembly PCR using six short primers ranging in lengths from 47 to 59 nt to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. The PCR cycle numbers for steps 1, 2 and 3 were 5, 10 and 20, respectively. The expected amplicon lengths of each step (88, 162 and 198 bp, respectively) are indicated above the lanes. Primer dimer bands can be observed below the main product bands. M, DNA marker.

    Techniques Used: Polymerase Cycling Assembly, Sequencing, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Amplification, Marker

    Biochemical characterization of Dpo4-5m. ( a ) Chemically synthesized and purified 40.8 kDa Dpo4-5m, as well as recombinant Dpo4-5m (with His 6 tag) purified from the E. coli strain BL21(DE3) were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained by Coomassie Brilliant Blue. A small fraction of unligated peptide segments can be observed in the synthetic Dpo4-5m. M, protein marker. ( b ) PCR amplification of a 200 bp sequence by recombinant wild-type Dpo4 ('WT Dpo4'), recombinant Dpo4-5m ('Recombinant') and synthetic Dpo4-5m ('Synthetic'), performed in 50 m M HEPES (pH 7.5), 5 m M MgCl 2 , 50 m M NaCl, 0.1 m M EDTA, 5 m M dithiothreitol, 10% glycerol, 3% dimethyl sulfoxide, 0.1 mg ml −1 bovine serum albumin, 200 μ M (each) ultrapure dNTPs, 0.5 μ M (each) primers, 2 n M linearized dsDNA template and ~300 n M polymerase for 35 cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. NC, negative control with template and primers but without enzyme. ( c ) PCR amplification of a 200 bp sequence by synthetic Dpo4-5m, sampled from multiple cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView, with cycle numbers from which they were sampled indicated above the lanes. M, DNA marker.
    Figure Legend Snippet: Biochemical characterization of Dpo4-5m. ( a ) Chemically synthesized and purified 40.8 kDa Dpo4-5m, as well as recombinant Dpo4-5m (with His 6 tag) purified from the E. coli strain BL21(DE3) were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained by Coomassie Brilliant Blue. A small fraction of unligated peptide segments can be observed in the synthetic Dpo4-5m. M, protein marker. ( b ) PCR amplification of a 200 bp sequence by recombinant wild-type Dpo4 ('WT Dpo4'), recombinant Dpo4-5m ('Recombinant') and synthetic Dpo4-5m ('Synthetic'), performed in 50 m M HEPES (pH 7.5), 5 m M MgCl 2 , 50 m M NaCl, 0.1 m M EDTA, 5 m M dithiothreitol, 10% glycerol, 3% dimethyl sulfoxide, 0.1 mg ml −1 bovine serum albumin, 200 μ M (each) ultrapure dNTPs, 0.5 μ M (each) primers, 2 n M linearized dsDNA template and ~300 n M polymerase for 35 cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. NC, negative control with template and primers but without enzyme. ( c ) PCR amplification of a 200 bp sequence by synthetic Dpo4-5m, sampled from multiple cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView, with cycle numbers from which they were sampled indicated above the lanes. M, DNA marker.

    Techniques Used: Synthesized, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Marker, Polymerase Chain Reaction, Amplification, Sequencing, Agarose Gel Electrophoresis, Negative Control

    9) Product Images from "Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens"

    Article Title: Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens

    Journal: bioRxiv

    doi: 10.1101/842625

    Quantification for optimizing the VFD rotational speed for fDNA yields. After proteinase K and VFD treatment at the indicated speeds, (A) absorbance at 260 nm and (B) the ratio of absorbances 260:280 nm quantifies DNA and protein yields, respectively, with positive and negative controls. Full UV-vis spectra for these samples are shown in S3 Fig. SYBR Green I fluorescence-quantified (C) dsDNA concentration and (D) fold increase in dsDNA yield between non-VFD-processed and VFD-processed samples. The negative control indicates samples not subjected to VFD processing. Buffer only controls lacked lobster tissue. The positive controls included DNA that had not been formalin fixed. Additional controls demonstrated a conventional method and an intermediate method (processing time of the conventional method and temperature of the VFD-mediated method) used to process fixed and fresh tissue without the VFD. The error bars designate the standard deviation for sample measurements at the indicated condition (technical replicates, n = 3).
    Figure Legend Snippet: Quantification for optimizing the VFD rotational speed for fDNA yields. After proteinase K and VFD treatment at the indicated speeds, (A) absorbance at 260 nm and (B) the ratio of absorbances 260:280 nm quantifies DNA and protein yields, respectively, with positive and negative controls. Full UV-vis spectra for these samples are shown in S3 Fig. SYBR Green I fluorescence-quantified (C) dsDNA concentration and (D) fold increase in dsDNA yield between non-VFD-processed and VFD-processed samples. The negative control indicates samples not subjected to VFD processing. Buffer only controls lacked lobster tissue. The positive controls included DNA that had not been formalin fixed. Additional controls demonstrated a conventional method and an intermediate method (processing time of the conventional method and temperature of the VFD-mediated method) used to process fixed and fresh tissue without the VFD. The error bars designate the standard deviation for sample measurements at the indicated condition (technical replicates, n = 3).

    Techniques Used: SYBR Green Assay, Fluorescence, Concentration Assay, Negative Control, Standard Deviation

    Amplification of an 183-bp fDNA target from the ATP synthase gene of the lobster mitochondrial genome. (A) Quantitative PCR and (B) agarose DNA gel electrophoresis identified 7 krpm as the optimal VFD rotational speed for qPCR amplification. Threshold cycle and endpoint fluorescence values are provided in S2 Table. The variable-rotational speed PCR reactions were compared to a no template control (NTC), a fresh lobster DNA positive control (+), and a non-VFD-processed negative control (–). (C) The 7 krpm VFD-processed qPCR product (*) was subjected to Sanger sequencing; a mutation (G2728A, GenBank No. HQ402925 ) was observed (highlighted).
    Figure Legend Snippet: Amplification of an 183-bp fDNA target from the ATP synthase gene of the lobster mitochondrial genome. (A) Quantitative PCR and (B) agarose DNA gel electrophoresis identified 7 krpm as the optimal VFD rotational speed for qPCR amplification. Threshold cycle and endpoint fluorescence values are provided in S2 Table. The variable-rotational speed PCR reactions were compared to a no template control (NTC), a fresh lobster DNA positive control (+), and a non-VFD-processed negative control (–). (C) The 7 krpm VFD-processed qPCR product (*) was subjected to Sanger sequencing; a mutation (G2728A, GenBank No. HQ402925 ) was observed (highlighted).

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, DNA Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Positive Control, Negative Control, Sequencing, Mutagenesis

    Schematic of the VFD-mediated fDNA recovery technique. (A) The protocol begins with Vortex Fluidic Device (VFD) treatment (7 krpm, room temperature, abbreviated RT, 1 h) of a mixture of proteinase K and the frozen, then broken-up tissue. The reaction mixture is next processed to remove solids and DNA polymerase inhibitors. The recovered fDNA is then purified and concentrated. Finally, the DNA is amplified, quantified, and characterized by (B) qPCR and (C) DNA sequencing of the samples. Larger versions of panels B and C are provided in S1 and S2 Figs. Threshold cycle (C t ) and endpoint fluorescence values are given in S1 Table.
    Figure Legend Snippet: Schematic of the VFD-mediated fDNA recovery technique. (A) The protocol begins with Vortex Fluidic Device (VFD) treatment (7 krpm, room temperature, abbreviated RT, 1 h) of a mixture of proteinase K and the frozen, then broken-up tissue. The reaction mixture is next processed to remove solids and DNA polymerase inhibitors. The recovered fDNA is then purified and concentrated. Finally, the DNA is amplified, quantified, and characterized by (B) qPCR and (C) DNA sequencing of the samples. Larger versions of panels B and C are provided in S1 and S2 Figs. Threshold cycle (C t ) and endpoint fluorescence values are given in S1 Table.

    Techniques Used: Purification, Amplification, Real-time Polymerase Chain Reaction, DNA Sequencing, Fluorescence

    10) Product Images from "Evidence for feasibility of fetal trophoblastic cell‐based noninvasive prenatal testing †) Evidence for feasibility of fetal trophoblastic cell‐based noninvasive prenatal testing"

    Article Title: Evidence for feasibility of fetal trophoblastic cell‐based noninvasive prenatal testing †) Evidence for feasibility of fetal trophoblastic cell‐based noninvasive prenatal testing

    Journal: Prenatal Diagnosis

    doi: 10.1002/pd.4924

    Multiple fetal trophoblastic cells isolated from one patient. A. Eight fetal cells (confirmed by STR analysis) from a single patient (subject 365) showing the range of nuclear (DAPI; blue) and cytokeratin (CK; green) staining morphology. B. Two of the fetal cells from subject 365 demonstrating uniform (top right) and fragmented (bottom right) DAPI staining. C. Hemi‐nested Y‐chromosome specific PCR performed on the eight fetal cells isolated from subject 365 (male fetus), showing positive amplification of one or more targeted regions on the Y chromosome (SRY, DYS14, DAZ). Numbers correspond to the number prefix for each individual cell in A. D. Ampli 1 STR analysis comparing WGA product from a single fetal cell (G648, subject 365) and genomic DNA from each parent. The three loci shown here demonstrate the expected paternal inheritance (arrows) from the Y chromosome (SRY; left panel) and bi‐parental inheritance from chromosomes 5 (D5S818; center panel) and 18 (D18S535; right panel)
    Figure Legend Snippet: Multiple fetal trophoblastic cells isolated from one patient. A. Eight fetal cells (confirmed by STR analysis) from a single patient (subject 365) showing the range of nuclear (DAPI; blue) and cytokeratin (CK; green) staining morphology. B. Two of the fetal cells from subject 365 demonstrating uniform (top right) and fragmented (bottom right) DAPI staining. C. Hemi‐nested Y‐chromosome specific PCR performed on the eight fetal cells isolated from subject 365 (male fetus), showing positive amplification of one or more targeted regions on the Y chromosome (SRY, DYS14, DAZ). Numbers correspond to the number prefix for each individual cell in A. D. Ampli 1 STR analysis comparing WGA product from a single fetal cell (G648, subject 365) and genomic DNA from each parent. The three loci shown here demonstrate the expected paternal inheritance (arrows) from the Y chromosome (SRY; left panel) and bi‐parental inheritance from chromosomes 5 (D5S818; center panel) and 18 (D18S535; right panel)

    Techniques Used: Isolation, Staining, Polymerase Chain Reaction, Amplification, Whole Genome Amplification

    11) Product Images from "Regulation of Anti-Plasmodium Immunity by a LITAF-like Transcription Factor in the Malaria Vector Anopheles gambiae"

    Article Title: Regulation of Anti-Plasmodium Immunity by a LITAF-like Transcription Factor in the Malaria Vector Anopheles gambiae

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002965

    PCR-assisted DNA-binding site selection reveals consensus LL3 DNA-binding motifs. (A) Experimental outline of the two methods [“cold” (non-radioactive) or “hot” (radioactive)] used to obtain consensus DNA binding sites for rLL3 by PCR-assisted DNA-binding site selection. Consensus motifs obtained from the “cold” method using a 10 bp degenerate sequence (B) or from the 20 bp degenerate sequences recovered using the “hot” method (C) are shown to the right. All recovered sequences used as input to generate the consensus motifs are listed in Table S2 and all motifs generated by the MEME program are displayed in Figure S4 . EMSA: Electrophoretic Mobility-Shift Assay.
    Figure Legend Snippet: PCR-assisted DNA-binding site selection reveals consensus LL3 DNA-binding motifs. (A) Experimental outline of the two methods [“cold” (non-radioactive) or “hot” (radioactive)] used to obtain consensus DNA binding sites for rLL3 by PCR-assisted DNA-binding site selection. Consensus motifs obtained from the “cold” method using a 10 bp degenerate sequence (B) or from the 20 bp degenerate sequences recovered using the “hot” method (C) are shown to the right. All recovered sequences used as input to generate the consensus motifs are listed in Table S2 and all motifs generated by the MEME program are displayed in Figure S4 . EMSA: Electrophoretic Mobility-Shift Assay.

    Techniques Used: Polymerase Chain Reaction, Binding Assay, Selection, Sequencing, Generated, Electrophoretic Mobility Shift Assay

    12) Product Images from "Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction"

    Article Title: Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.8

    PCR amplification of various sequences by synthetic Dpo4-5m. ( a ) PCR amplification of sequences with lengths ranging from 110 bp to 1 kb by synthetic Dpo4-5m for 35 cycles. The expected amplicon lengths are indicated above the lanes. Primer dimer bands can be observed below the main product bands. The extension time was set to 2 min per cycle for 110–300 bp sequences, 5 min for 400–600 bp sequences and 10 min for 700–1000 bp sequences. W/o template: negative control with primers and enzyme but without template; NC (110 bp)/NC (1 kb): negative control with 110 bp or 1 kb templates and primers but without enzyme. ( b ) PCR amplification of the 1.1 kb dpo4 gene by synthetic Dpo4-5 m for 35 cycles, with the extension time set to 10 min per cycle. ( c ) PCR amplification of the 120 bp 5S E. coli rRNA gene rrfB by synthetic Dpo4-5m for 35 cycles, with the extension time set to 2 min per cycle. ( d ) PCR amplification of the 1.5 kb 16S E. coli rRNA gene rrsC by synthetic Dpo4-5m for 35 cycles, with the extension time set to 15 min per cycle. A primer dimer band can be observed below the main product band. All the PCR products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. The expected amplicon lengths are indicated above the lanes. NC, negative control with template and primers but without enzyme. M, DNA marker.
    Figure Legend Snippet: PCR amplification of various sequences by synthetic Dpo4-5m. ( a ) PCR amplification of sequences with lengths ranging from 110 bp to 1 kb by synthetic Dpo4-5m for 35 cycles. The expected amplicon lengths are indicated above the lanes. Primer dimer bands can be observed below the main product bands. The extension time was set to 2 min per cycle for 110–300 bp sequences, 5 min for 400–600 bp sequences and 10 min for 700–1000 bp sequences. W/o template: negative control with primers and enzyme but without template; NC (110 bp)/NC (1 kb): negative control with 110 bp or 1 kb templates and primers but without enzyme. ( b ) PCR amplification of the 1.1 kb dpo4 gene by synthetic Dpo4-5 m for 35 cycles, with the extension time set to 10 min per cycle. ( c ) PCR amplification of the 120 bp 5S E. coli rRNA gene rrfB by synthetic Dpo4-5m for 35 cycles, with the extension time set to 2 min per cycle. ( d ) PCR amplification of the 1.5 kb 16S E. coli rRNA gene rrsC by synthetic Dpo4-5m for 35 cycles, with the extension time set to 15 min per cycle. A primer dimer band can be observed below the main product band. All the PCR products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. The expected amplicon lengths are indicated above the lanes. NC, negative control with template and primers but without enzyme. M, DNA marker.

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control, Agarose Gel Electrophoresis, Staining, Marker

    Assembly PCR by synthetic Dpo4-5m. ( a , b ) Assembly PCR using two long primers ( tC19Z -F115 and tC19Z -R113) with a 30 bp sequence overlap for 20 cycles to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. Exo I: treated by exonuclease I (which digests single-stranded DNA (ssDNA) but not dsDNA). ( c , d ) Three-step assembly PCR using six short primers ranging in lengths from 47 to 59 nt to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. The PCR cycle numbers for steps 1, 2 and 3 were 5, 10 and 20, respectively. The expected amplicon lengths of each step (88, 162 and 198 bp, respectively) are indicated above the lanes. Primer dimer bands can be observed below the main product bands. M, DNA marker.
    Figure Legend Snippet: Assembly PCR by synthetic Dpo4-5m. ( a , b ) Assembly PCR using two long primers ( tC19Z -F115 and tC19Z -R113) with a 30 bp sequence overlap for 20 cycles to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. Exo I: treated by exonuclease I (which digests single-stranded DNA (ssDNA) but not dsDNA). ( c , d ) Three-step assembly PCR using six short primers ranging in lengths from 47 to 59 nt to obtain the 198 bp tC19Z gene, analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView. The PCR cycle numbers for steps 1, 2 and 3 were 5, 10 and 20, respectively. The expected amplicon lengths of each step (88, 162 and 198 bp, respectively) are indicated above the lanes. Primer dimer bands can be observed below the main product bands. M, DNA marker.

    Techniques Used: Polymerase Cycling Assembly, Sequencing, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Amplification, Marker

    Biochemical characterization of Dpo4-5m. ( a ) Chemically synthesized and purified 40.8 kDa Dpo4-5m, as well as recombinant Dpo4-5m (with His 6 tag) purified from the E. coli strain BL21(DE3) were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained by Coomassie Brilliant Blue. A small fraction of unligated peptide segments can be observed in the synthetic Dpo4-5m. M, protein marker. ( b ) PCR amplification of a 200 bp sequence by recombinant wild-type Dpo4 ('WT Dpo4'), recombinant Dpo4-5m ('Recombinant') and synthetic Dpo4-5m ('Synthetic'), performed in 50 m M HEPES (pH 7.5), 5 m M MgCl 2 , 50 m M NaCl, 0.1 m M EDTA, 5 m M dithiothreitol, 10% glycerol, 3% dimethyl sulfoxide, 0.1 mg ml −1 bovine serum albumin, 200 μ M (each) ultrapure dNTPs, 0.5 μ M (each) primers, 2 n M linearized dsDNA template and ~300 n M polymerase for 35 cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. NC, negative control with template and primers but without enzyme. ( c ) PCR amplification of a 200 bp sequence by synthetic Dpo4-5m, sampled from multiple cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView, with cycle numbers from which they were sampled indicated above the lanes. M, DNA marker.
    Figure Legend Snippet: Biochemical characterization of Dpo4-5m. ( a ) Chemically synthesized and purified 40.8 kDa Dpo4-5m, as well as recombinant Dpo4-5m (with His 6 tag) purified from the E. coli strain BL21(DE3) were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained by Coomassie Brilliant Blue. A small fraction of unligated peptide segments can be observed in the synthetic Dpo4-5m. M, protein marker. ( b ) PCR amplification of a 200 bp sequence by recombinant wild-type Dpo4 ('WT Dpo4'), recombinant Dpo4-5m ('Recombinant') and synthetic Dpo4-5m ('Synthetic'), performed in 50 m M HEPES (pH 7.5), 5 m M MgCl 2 , 50 m M NaCl, 0.1 m M EDTA, 5 m M dithiothreitol, 10% glycerol, 3% dimethyl sulfoxide, 0.1 mg ml −1 bovine serum albumin, 200 μ M (each) ultrapure dNTPs, 0.5 μ M (each) primers, 2 n M linearized dsDNA template and ~300 n M polymerase for 35 cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView. NC, negative control with template and primers but without enzyme. ( c ) PCR amplification of a 200 bp sequence by synthetic Dpo4-5m, sampled from multiple cycles. The products were analyzed by 2% agarose gel electrophoresis and stained by GoldView, with cycle numbers from which they were sampled indicated above the lanes. M, DNA marker.

    Techniques Used: Synthesized, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Marker, Polymerase Chain Reaction, Amplification, Sequencing, Agarose Gel Electrophoresis, Negative Control

    13) Product Images from "Loci specific epigenetic drug sensitivity"

    Article Title: Loci specific epigenetic drug sensitivity

    Journal: bioRxiv

    doi: 10.1101/686139

    Combinatorial pooling massively and parallely identify barcode of individual clones. (a) The combinatorial pooled sequencing involves encoding and decoding steps. Each individual clone was split into 4 out of 18 pooled samples according to the designs. Genomic DNA from each pooled sample was extracted and barcodes were amplified and labeled with 6-bp sample index. Amplicons from all samples were mixed together and prepared for NGS. Detected barcodes were deconvoluted to match barcode ID with clone ID. (b) The expression distribution of mClover protein was measured by high-throughput flow cytometer and displayed by stacked probability density function. Each row in the heatmap represents a single histogram from a single clonal line with the probability density function colorcoded. (c) A scatter plot of reporter expression noise, measured as log-transformed squared coefficient of variation (CV 2 ), and mClover mean across all examined positions affirms unique chromatin environments across the genome.
    Figure Legend Snippet: Combinatorial pooling massively and parallely identify barcode of individual clones. (a) The combinatorial pooled sequencing involves encoding and decoding steps. Each individual clone was split into 4 out of 18 pooled samples according to the designs. Genomic DNA from each pooled sample was extracted and barcodes were amplified and labeled with 6-bp sample index. Amplicons from all samples were mixed together and prepared for NGS. Detected barcodes were deconvoluted to match barcode ID with clone ID. (b) The expression distribution of mClover protein was measured by high-throughput flow cytometer and displayed by stacked probability density function. Each row in the heatmap represents a single histogram from a single clonal line with the probability density function colorcoded. (c) A scatter plot of reporter expression noise, measured as log-transformed squared coefficient of variation (CV 2 ), and mClover mean across all examined positions affirms unique chromatin environments across the genome.

    Techniques Used: Sequencing, Amplification, Labeling, Next-Generation Sequencing, Expressing, High Throughput Screening Assay, Flow Cytometry, Transformation Assay

    14) Product Images from "Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis"

    Article Title: Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

    Journal: Microbial biotechnology

    doi: 10.1111/j.1751-7915.2010.00230.x

    PCR‐based gene mutagenesis and plasmid multimerization. A. Relevant features of the vector pNWP43N‐Bscel5. P 43 , SP nprB , Bscel5 and term represent the P 43 promoter, the NprB signal peptide‐encoding sequence, gene of family 5 endoglucanse and terminator of Bscel5 from B. subtilis respectively. ColE1 ori , repB and cat represent the sequences coding for the ColE1 replication origin, replicase and chloramphenicol resistance marker respectively. The arrows show the transcription directions for these genes. B. The flow scheme of the two‐step PCR procedure for the gene mutagenesis and plasmid multimerization. gh5 , family 5 glycoside hydrolase‐encoding sequence; cbm3 , family 3 carbohydrate‐binding module‐encoding sequence. P1, P2, P3 and P4 denote the positions of the primers for the PCR amplification. This figure was not drawn to scale. C. Plasmid multimerization by PCR. Lanes: M, DNA markers; 1, PCR‐linearized pNWP43N‐Bscel5; 2, error‐prone PCR product of SPnprB‐Bscel5 ; 3, multimerized plasmid; 4, multimer digested with PstI/HindIII.
    Figure Legend Snippet: PCR‐based gene mutagenesis and plasmid multimerization. A. Relevant features of the vector pNWP43N‐Bscel5. P 43 , SP nprB , Bscel5 and term represent the P 43 promoter, the NprB signal peptide‐encoding sequence, gene of family 5 endoglucanse and terminator of Bscel5 from B. subtilis respectively. ColE1 ori , repB and cat represent the sequences coding for the ColE1 replication origin, replicase and chloramphenicol resistance marker respectively. The arrows show the transcription directions for these genes. B. The flow scheme of the two‐step PCR procedure for the gene mutagenesis and plasmid multimerization. gh5 , family 5 glycoside hydrolase‐encoding sequence; cbm3 , family 3 carbohydrate‐binding module‐encoding sequence. P1, P2, P3 and P4 denote the positions of the primers for the PCR amplification. This figure was not drawn to scale. C. Plasmid multimerization by PCR. Lanes: M, DNA markers; 1, PCR‐linearized pNWP43N‐Bscel5; 2, error‐prone PCR product of SPnprB‐Bscel5 ; 3, multimerized plasmid; 4, multimer digested with PstI/HindIII.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Plasmid Preparation, Sequencing, Marker, Flow Cytometry, Binding Assay, Amplification

    15) Product Images from "Various Mechanisms Involve the Nuclear Factor (Erythroid-Derived 2)-Like (NRF2) to Achieve Cytoprotection in Long-Term Cisplatin-Treated Urothelial Carcinoma Cell Lines"

    Article Title: Various Mechanisms Involve the Nuclear Factor (Erythroid-Derived 2)-Like (NRF2) to Achieve Cytoprotection in Long-Term Cisplatin-Treated Urothelial Carcinoma Cell Lines

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18081680

    NRF2 knockdown sensitises LTTs towards cisplatin by increasing DNA damage. NRF2 protein ( a ) and mRNA ( b ) expression was measured in LTTs after siNRF2-mediated knockdown and their negative controls. As a loading control, α-Tubulin was detected. GSR, NQO1, HMOX1, GPX1, GSTP1, and p62/SQSTM1 mRNA expression in siNRF2-transfected ( c ) RT-112-LTT, ( d ) J82-LTT, ( e ) 253J-LTT, ( f ) T-24-LTT and their negative controls were measured by qRT-PCR. Expression levels in the negative control were set as 1. SDHA mRNA was used as a reference and relative expression was calculated by the 2 −ΔΔ C t method. ( g ) Cell viability was measured 72 h after cisplatin treatment by CellTiterGlo assay in LTTs transfected with siRNA targeting NRF2 or a non-targeting negative control siRNA. ( h ) Quantification of pH2A.X Ser139 foci by immunofluorescent staining in LTTs transfected with siRNA targeting NRF2 or a non-targeting negative control siRNA and treated or not treated with cisplatin after 72 h. Values represent the mean ± SD of biological duplicates, * p
    Figure Legend Snippet: NRF2 knockdown sensitises LTTs towards cisplatin by increasing DNA damage. NRF2 protein ( a ) and mRNA ( b ) expression was measured in LTTs after siNRF2-mediated knockdown and their negative controls. As a loading control, α-Tubulin was detected. GSR, NQO1, HMOX1, GPX1, GSTP1, and p62/SQSTM1 mRNA expression in siNRF2-transfected ( c ) RT-112-LTT, ( d ) J82-LTT, ( e ) 253J-LTT, ( f ) T-24-LTT and their negative controls were measured by qRT-PCR. Expression levels in the negative control were set as 1. SDHA mRNA was used as a reference and relative expression was calculated by the 2 −ΔΔ C t method. ( g ) Cell viability was measured 72 h after cisplatin treatment by CellTiterGlo assay in LTTs transfected with siRNA targeting NRF2 or a non-targeting negative control siRNA. ( h ) Quantification of pH2A.X Ser139 foci by immunofluorescent staining in LTTs transfected with siRNA targeting NRF2 or a non-targeting negative control siRNA and treated or not treated with cisplatin after 72 h. Values represent the mean ± SD of biological duplicates, * p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Negative Control, Staining

    16) Product Images from "Loci specific epigenetic drug sensitivity"

    Article Title: Loci specific epigenetic drug sensitivity

    Journal: bioRxiv

    doi: 10.1101/686139

    Diverse insertion landscapes of barcoded reporter. (a) Reporter mapping by inverse PCR. Genomic DNA of founder cells was extracted, digested with restriction enzyme MspI and self-ligated to stitch barcode with its neighboring genome. Ligated product was amplified and followed by next generation sequencing. (b) Ideogram plot displaying reporter integration sites of individual clones in the library. Centromere position is indicated in red and stalk is marked in light blue. Heterochromatic region, which tend to be rich with adenine and thymine and relatively gene-poor, is represented by black and variation of grey. R-band in white on the ideogram is less condensed chromatin that is transcriptionally more active.
    Figure Legend Snippet: Diverse insertion landscapes of barcoded reporter. (a) Reporter mapping by inverse PCR. Genomic DNA of founder cells was extracted, digested with restriction enzyme MspI and self-ligated to stitch barcode with its neighboring genome. Ligated product was amplified and followed by next generation sequencing. (b) Ideogram plot displaying reporter integration sites of individual clones in the library. Centromere position is indicated in red and stalk is marked in light blue. Heterochromatic region, which tend to be rich with adenine and thymine and relatively gene-poor, is represented by black and variation of grey. R-band in white on the ideogram is less condensed chromatin that is transcriptionally more active.

    Techniques Used: Inverse PCR, Amplification, Next-Generation Sequencing, Clone Assay

    Chromosomal position effects 5 Azacytidine sensitivity through DNA methylation-independent mechanism. (a) DNA methylation profiles of selected clones were simultaneously revealed by bisulfite conversion, targeted PCR and deep sequencing. (b) Examples of mClover distribution from clones with reduced expression (top row) and induced (bottom row) to 5 Azacytidine. (c) Locus-specific bisulfite sequencing of the CMV promoters of representative clones exhibiting hyposensitivity and hypersensitivity to 5 Azacytidine.
    Figure Legend Snippet: Chromosomal position effects 5 Azacytidine sensitivity through DNA methylation-independent mechanism. (a) DNA methylation profiles of selected clones were simultaneously revealed by bisulfite conversion, targeted PCR and deep sequencing. (b) Examples of mClover distribution from clones with reduced expression (top row) and induced (bottom row) to 5 Azacytidine. (c) Locus-specific bisulfite sequencing of the CMV promoters of representative clones exhibiting hyposensitivity and hypersensitivity to 5 Azacytidine.

    Techniques Used: DNA Methylation Assay, Clone Assay, Polymerase Chain Reaction, Sequencing, Expressing, Methylation Sequencing

    17) Product Images from "RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression"

    Article Title: RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00105-19

    Single-copy loss of RB1 can create cancer-enabling phenotypes. (A) H E staining of lung tissue with the highest number of RB1 +/− seeding events. (B) QuPath coloring to denote tumor tissue within these lungs (red). (C) DNA was extracted from nodules of tumor cells in paraffin-embedded tissue containing these RB1 +/− cells or an RB1 +/+ control. PCR was performed to amplify exon 22 from recovered DNA and controls isolated from cell culture to verify the final genotype of cells in this sample. (D) The 10 most prevalent cancers were analyzed for RB1 gene alterations using TCGA and Pan-Cancer Atlas data, using cBioPortal. A deep deletion is consistent with biallelic loss of RB1 , whereas a shallow deletion is suggestive of heterozygous RB1 deletion.
    Figure Legend Snippet: Single-copy loss of RB1 can create cancer-enabling phenotypes. (A) H E staining of lung tissue with the highest number of RB1 +/− seeding events. (B) QuPath coloring to denote tumor tissue within these lungs (red). (C) DNA was extracted from nodules of tumor cells in paraffin-embedded tissue containing these RB1 +/− cells or an RB1 +/+ control. PCR was performed to amplify exon 22 from recovered DNA and controls isolated from cell culture to verify the final genotype of cells in this sample. (D) The 10 most prevalent cancers were analyzed for RB1 gene alterations using TCGA and Pan-Cancer Atlas data, using cBioPortal. A deep deletion is consistent with biallelic loss of RB1 , whereas a shallow deletion is suggestive of heterozygous RB1 deletion.

    Techniques Used: Staining, Polymerase Chain Reaction, Isolation, Cell Culture

    18) Product Images from "Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae"

    Article Title: Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt891

    MS identifies proteins co-purifying with the single copy PHO5 gene. ( A ) DNA analysis of samples of a TAP of PHO5 gene rings. Yeast strain y2629 carrying a PHO5 locus flanked by RS elements was subjected to TAP. DNA was isolated from samples CE, SUP, P and FT, E, and B from IgG and calmodulin affinity purifications, digested with NcoI and analyzed as described in the legend to Figure 1 C. Chromatin was purified from 1.5 × 10 12 cells and 0.01% (CE), 0.02% (SUP, P, FT), 2.5% (B, E; IgG, FT; calmodulin) and 3.3% (B, E, lanes 8–9; calmodulin) from the respective sample were analyzed. Positions of DNA size markers and of the NcoI fragment of the PHO5 gene ring are indicated. ( B ) Analysis of proteins co-purifying with PHO5 chromatin domains. Proteins co-purifying from calmodulin sepharose with LexA-CBP from yeast strains y2629 ( PHO5 , see above) and y2628 (control) carrying an unmodified PHO5 locus were separated in an SDS–PAGE gradient gel (4–12%) stained with silver. The LexA bait protein was purified from ∼1.5 × 10 12 cells, and 95% of the respective samples were used for analysis. For relative quantification, defined amounts of recombinant human histone octamers were analyzed in the same gel. Positions of protein size markers, and LexA-CBP, the core histones, as well as the ring DNA (visualized by silver stain) are indicated. ( C ) Graphical summary of enrichment of proteins in PHO5 gene ring purifications. Proteins co-purifying with PHO5 gene rings isolated from cells in which PHO5 transcription was repressed (y464[K2048]), or constitutively activated (y465[K2048]) were subjected to iTRAQ analysis in direct comparison with purifications of the corresponding control strains (y454[K2048], or y455[K2048], respectively). Data of two independent replicates were evaluated as described in the legend to Figure 3 .
    Figure Legend Snippet: MS identifies proteins co-purifying with the single copy PHO5 gene. ( A ) DNA analysis of samples of a TAP of PHO5 gene rings. Yeast strain y2629 carrying a PHO5 locus flanked by RS elements was subjected to TAP. DNA was isolated from samples CE, SUP, P and FT, E, and B from IgG and calmodulin affinity purifications, digested with NcoI and analyzed as described in the legend to Figure 1 C. Chromatin was purified from 1.5 × 10 12 cells and 0.01% (CE), 0.02% (SUP, P, FT), 2.5% (B, E; IgG, FT; calmodulin) and 3.3% (B, E, lanes 8–9; calmodulin) from the respective sample were analyzed. Positions of DNA size markers and of the NcoI fragment of the PHO5 gene ring are indicated. ( B ) Analysis of proteins co-purifying with PHO5 chromatin domains. Proteins co-purifying from calmodulin sepharose with LexA-CBP from yeast strains y2629 ( PHO5 , see above) and y2628 (control) carrying an unmodified PHO5 locus were separated in an SDS–PAGE gradient gel (4–12%) stained with silver. The LexA bait protein was purified from ∼1.5 × 10 12 cells, and 95% of the respective samples were used for analysis. For relative quantification, defined amounts of recombinant human histone octamers were analyzed in the same gel. Positions of protein size markers, and LexA-CBP, the core histones, as well as the ring DNA (visualized by silver stain) are indicated. ( C ) Graphical summary of enrichment of proteins in PHO5 gene ring purifications. Proteins co-purifying with PHO5 gene rings isolated from cells in which PHO5 transcription was repressed (y464[K2048]), or constitutively activated (y465[K2048]) were subjected to iTRAQ analysis in direct comparison with purifications of the corresponding control strains (y454[K2048], or y455[K2048], respectively). Data of two independent replicates were evaluated as described in the legend to Figure 3 .

    Techniques Used: Mass Spectrometry, Isolation, Purification, SDS Page, Staining, Recombinant, Silver Staining

    Distinct domains of the rDNA locus can be purified from yeast. ( A ) Purification of chromosomal domains. LEXA, cluster of LexA DNA binding sites; RS, sequences for site specific recombination; LexA-TAP, recombinant LexA fusion protein; filled ovals, chromatin components; filled circle, IgG coated magnetic beads. ( B ) Genetic manipulation of the rDNA locus. The rDNA locus on chromosome XII is depicted on the top. CEN, centromere; TEL, telomere; 35S, 25S, 18S, 5S rRNA coding regions; IGS, intergenic spacer region; ARS, autonomous replication sequence (gray circles); E-pro, expansion promoter; black arrows, transcription start sites used by Pol I, II, III; gray arrows; insertion sites of RS elements and LexA DNA binding sites; numbers in parentheses represent sizes of the different domains. ( C ) DNA analysis of samples of a chromatin domain purification and of a control purification. Yeast strains y2381 (35S) and y2378 (control), carrying the 35S rRNA gene domain flanked by RS sites, or lacking recombination sites, respectively, were subjected to the purification procedure. DNA was isolated from samples CE, SUP, P, FT, E and B ( Figure 1 A), SacII digested and analyzed in a 1% agarose gel stained with SYBR® Safe (Life technologies). The LexA bait protein was purified from 10 11 cells and 0.7% (CE, P), 2.5% (SUP, FT) and 8.3% (E, B) from the respective samples were analyzed. Positions of DNA size markers, and of the linearized 35S rRNA gene ring DNA (35S) are indicated. ( D ) DNA analysis of purified rDNA chromatin domains. Chromatin domains were purified from yeast strains y2384 (E-Pro), y2379 (5S), y2383 (ARS), y2380 (18S), y2381 (35S) and y2382 (rDNA). DNA was isolated from TEV eluates, digested with NcoI (E-pro, 5S, ARS) or SacII (18S, 35S, rDNA) and analyzed as described in the legend to Figure 1 A. The LexA bait protein was purified from 10 11 cells, and 5% of the respective samples were analyzed. Positions of DNA size markers and of restriction fragments of the individual rDNA segments are indicated. Asterisks mark restriction fragments from higher-order recombination products of the respective domain.
    Figure Legend Snippet: Distinct domains of the rDNA locus can be purified from yeast. ( A ) Purification of chromosomal domains. LEXA, cluster of LexA DNA binding sites; RS, sequences for site specific recombination; LexA-TAP, recombinant LexA fusion protein; filled ovals, chromatin components; filled circle, IgG coated magnetic beads. ( B ) Genetic manipulation of the rDNA locus. The rDNA locus on chromosome XII is depicted on the top. CEN, centromere; TEL, telomere; 35S, 25S, 18S, 5S rRNA coding regions; IGS, intergenic spacer region; ARS, autonomous replication sequence (gray circles); E-pro, expansion promoter; black arrows, transcription start sites used by Pol I, II, III; gray arrows; insertion sites of RS elements and LexA DNA binding sites; numbers in parentheses represent sizes of the different domains. ( C ) DNA analysis of samples of a chromatin domain purification and of a control purification. Yeast strains y2381 (35S) and y2378 (control), carrying the 35S rRNA gene domain flanked by RS sites, or lacking recombination sites, respectively, were subjected to the purification procedure. DNA was isolated from samples CE, SUP, P, FT, E and B ( Figure 1 A), SacII digested and analyzed in a 1% agarose gel stained with SYBR® Safe (Life technologies). The LexA bait protein was purified from 10 11 cells and 0.7% (CE, P), 2.5% (SUP, FT) and 8.3% (E, B) from the respective samples were analyzed. Positions of DNA size markers, and of the linearized 35S rRNA gene ring DNA (35S) are indicated. ( D ) DNA analysis of purified rDNA chromatin domains. Chromatin domains were purified from yeast strains y2384 (E-Pro), y2379 (5S), y2383 (ARS), y2380 (18S), y2381 (35S) and y2382 (rDNA). DNA was isolated from TEV eluates, digested with NcoI (E-pro, 5S, ARS) or SacII (18S, 35S, rDNA) and analyzed as described in the legend to Figure 1 A. The LexA bait protein was purified from 10 11 cells, and 5% of the respective samples were analyzed. Positions of DNA size markers and of restriction fragments of the individual rDNA segments are indicated. Asterisks mark restriction fragments from higher-order recombination products of the respective domain.

    Techniques Used: Purification, Binding Assay, Recombinant, Magnetic Beads, Sequencing, Isolation, Agarose Gel Electrophoresis, Staining

    Nucleosomal protection and nucleosome positions at 5S rDNA. ( A ) Restriction endonuclease accessibilities in purified and chromosomal 5S rDNA chromatin. Purified 5S rDNA chromatin and isolated nuclei from yeast strains y1997[K2049], and y2124 or y1599, respectively, were digested with increasing amounts of the indicated restriction enzymes (triangle on top of each pair of panels). DNA was isolated, digested with NcoI (chromatin ring) or PvuI/SphI (chromosome) and subjected to indirect endlabeling Southern blot analysis with the radioactively labeled probe 5S_2. Top: schematic representation of the 5S rRNA gene locus with restriction sites used to probe chromatin structure (black lines) and restriction sites of the secondary digestion (black arrows). The positions of uncut and cut fragments are shown on the right. The histogram shows the results of Southern blot quantification as a percentage of DNA cut at the highest restriction enzyme concentration. Average and standard deviations are from two independent biological replicates. ( B–D ) Determination of nucleosome positions at the 5S rRNA gene by single molecule EM analysis. (B) Isolated chromatin rings were subjected to psoralen crosslinking (black crosses). After DNA isolation, crosslinked molecules were relaxed with the nicking endonuclease Nt.AlwI, denatured and analyzed by EM under denaturing conditions. The panel on the right shows a representative electron micrograph. (C) After linearization with the restriction endonuclease NcoI before denaturation and EM analysis, 334 molecules were analyzed and categorized according to number (given on the left), size and position of the observed single-stranded bubbles. Representative electron micrographs for molecules of each class are shown. The percentage of each class in the total population of molecules is depicted in the lower right corner of the micrographs. The left-most electron micrographs show three classes containing a crosslinked 5S rRNA coding sequence (position indicated by a black bar in each electron micrograph). (D) The bar graph depicts bubble size distribution (in bp) of 701 single-stranded DNA bubbles measured in the total population of 334 molecules.
    Figure Legend Snippet: Nucleosomal protection and nucleosome positions at 5S rDNA. ( A ) Restriction endonuclease accessibilities in purified and chromosomal 5S rDNA chromatin. Purified 5S rDNA chromatin and isolated nuclei from yeast strains y1997[K2049], and y2124 or y1599, respectively, were digested with increasing amounts of the indicated restriction enzymes (triangle on top of each pair of panels). DNA was isolated, digested with NcoI (chromatin ring) or PvuI/SphI (chromosome) and subjected to indirect endlabeling Southern blot analysis with the radioactively labeled probe 5S_2. Top: schematic representation of the 5S rRNA gene locus with restriction sites used to probe chromatin structure (black lines) and restriction sites of the secondary digestion (black arrows). The positions of uncut and cut fragments are shown on the right. The histogram shows the results of Southern blot quantification as a percentage of DNA cut at the highest restriction enzyme concentration. Average and standard deviations are from two independent biological replicates. ( B–D ) Determination of nucleosome positions at the 5S rRNA gene by single molecule EM analysis. (B) Isolated chromatin rings were subjected to psoralen crosslinking (black crosses). After DNA isolation, crosslinked molecules were relaxed with the nicking endonuclease Nt.AlwI, denatured and analyzed by EM under denaturing conditions. The panel on the right shows a representative electron micrograph. (C) After linearization with the restriction endonuclease NcoI before denaturation and EM analysis, 334 molecules were analyzed and categorized according to number (given on the left), size and position of the observed single-stranded bubbles. Representative electron micrographs for molecules of each class are shown. The percentage of each class in the total population of molecules is depicted in the lower right corner of the micrographs. The left-most electron micrographs show three classes containing a crosslinked 5S rRNA coding sequence (position indicated by a black bar in each electron micrograph). (D) The bar graph depicts bubble size distribution (in bp) of 701 single-stranded DNA bubbles measured in the total population of 334 molecules.

    Techniques Used: Purification, Isolation, Southern Blot, Labeling, Concentration Assay, DNA Extraction, Sequencing

    19) Product Images from "An evolutionary arms race between KRAB zinc finger genes 91/93 and SVA/L1 retrotransposons"

    Article Title: An evolutionary arms race between KRAB zinc finger genes 91/93 and SVA/L1 retrotransposons

    Journal: Nature

    doi: 10.1038/nature13760

    KAP1 associates with recently emerged transposable elements a, Immunoblot incubated with anti-KAP1 antibody loaded with 1% input and eluates of KAP1-ChIP or IgG-ChIP derived from human ESC lysates. b , Diagram showing numbers of KAP1 peaks identified in two independent biological replicates and common peaks. c , Distribution of 9174 KAP1-ChIP-seq peaks over various DNA elements. d , Distribution of retrotransposon classes among KAP1-ChIP peaks from hESCs (left) or genome wide (right) e , KAP1 and H3K4me3 ChIP-seq and RNA-seq coverage tracks for a representative region on human chromosome 11 in hESCs (white-, grey-shaded) and TC11-mESCs (yellow-shaded). Blue arrows: de-repressed retrotransposons; black arrows: re-activated transcription; Red vertical shading: reactivated SVAs; orange shading: reactivated LTR12C. Blue and tan in RNA-seq tracks indicate positive and negative strand transcripts, respectively. Note that while the majority of SVAs display aberrant H3K4me3 signal, for unclear reasons not all SVAs display aberrant transcription in TC11-mESCs. sup: supernatant; Rep: biological replicate; TSS: transcription start site.
    Figure Legend Snippet: KAP1 associates with recently emerged transposable elements a, Immunoblot incubated with anti-KAP1 antibody loaded with 1% input and eluates of KAP1-ChIP or IgG-ChIP derived from human ESC lysates. b , Diagram showing numbers of KAP1 peaks identified in two independent biological replicates and common peaks. c , Distribution of 9174 KAP1-ChIP-seq peaks over various DNA elements. d , Distribution of retrotransposon classes among KAP1-ChIP peaks from hESCs (left) or genome wide (right) e , KAP1 and H3K4me3 ChIP-seq and RNA-seq coverage tracks for a representative region on human chromosome 11 in hESCs (white-, grey-shaded) and TC11-mESCs (yellow-shaded). Blue arrows: de-repressed retrotransposons; black arrows: re-activated transcription; Red vertical shading: reactivated SVAs; orange shading: reactivated LTR12C. Blue and tan in RNA-seq tracks indicate positive and negative strand transcripts, respectively. Note that while the majority of SVAs display aberrant H3K4me3 signal, for unclear reasons not all SVAs display aberrant transcription in TC11-mESCs. sup: supernatant; Rep: biological replicate; TSS: transcription start site.

    Techniques Used: Incubation, Chromatin Immunoprecipitation, Derivative Assay, Genome Wide, RNA Sequencing Assay

    20) Product Images from "A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly"

    Article Title: A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2016.08.001

    Gibson assembly of an endogenous tagging construct for FC1 [A ] Schematic of the plasmid, showing the 5′ UTR targeting segment ( 1 ; 5′ UTR), a segment ( 3 ) containing the blasticidin selection marker (BSD), alpha/beta tubulin intergenic rection (INTER), triple-Ty1 tag (3X Ty1), the first 500 bp of FC1 ( 2 ; FC1 Cod). and the endogenous tagging vector backbone for inducible expression (ET vector, 4 ). Sizes for each DNA segment are shown in parentheses. [ B ] Agarose gel showing the DNA segments used for Gibson assembly ( 1–4 , as labeled in A ), and the product ( P ) of the assembly digested with PacI and NsiI to show the completed endogenous tagging insert. The asterisk in lane 3 denotes the BLA-INTER-3X Ty1 DNA segment, which was excised from a previously-assembled construct by BamHI and HindIII restriction digest. The asterisk in lane 4 denotes the ET plasmid backbone, which was isolated by PacI and NsiI digest. [ C ] Cells containing the FC1 endogenous tagging construct were fixed and stained with an antibody against the flagella connector (FC; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.
    Figure Legend Snippet: Gibson assembly of an endogenous tagging construct for FC1 [A ] Schematic of the plasmid, showing the 5′ UTR targeting segment ( 1 ; 5′ UTR), a segment ( 3 ) containing the blasticidin selection marker (BSD), alpha/beta tubulin intergenic rection (INTER), triple-Ty1 tag (3X Ty1), the first 500 bp of FC1 ( 2 ; FC1 Cod). and the endogenous tagging vector backbone for inducible expression (ET vector, 4 ). Sizes for each DNA segment are shown in parentheses. [ B ] Agarose gel showing the DNA segments used for Gibson assembly ( 1–4 , as labeled in A ), and the product ( P ) of the assembly digested with PacI and NsiI to show the completed endogenous tagging insert. The asterisk in lane 3 denotes the BLA-INTER-3X Ty1 DNA segment, which was excised from a previously-assembled construct by BamHI and HindIII restriction digest. The asterisk in lane 4 denotes the ET plasmid backbone, which was isolated by PacI and NsiI digest. [ C ] Cells containing the FC1 endogenous tagging construct were fixed and stained with an antibody against the flagella connector (FC; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.

    Techniques Used: Construct, Plasmid Preparation, Selection, Marker, Expressing, Agarose Gel Electrophoresis, Labeling, Isolation, Staining, Fluorescence, Microscopy

    De novo assembly of a tagging construct for C-terminal triple-Ty1 tagging using Gibson assembly [A ] Schematic of the plasmid, showing the last 500 bp of the 4400 gene, which functions as a targeting segment ( 1 ; 4400 Cod), the triple-Ty1 tag ( 2; 3X Ty1), the alpha/beta tubulin intergenic region ( 3 ; INTER), the puromycin resistance gene ( 4 ; PAC), a 500 bp segment of the 3′ UTR of 4400 used for targeting ( 5 ; 3′ UTR), and the endogenous tagging vector backbone for inducible expression (ET vector, 6 ). Sizes for each DNA segment are shown in parentheses. [ B ] Agarose gel showing the DNA segments used for Gibson assembly ( 1–6 , as labeled in A ), and the product ( P ) of the assembly digested with PacI and NsiI to show the tagged insert. The asterisk in lane 6 denotes the plasmid backbone of the ET vector, which was used for the Gibson reaction. [ C ] Cells containing the 4400 endogenous tagging construct were fixed and stained with an antibody that detects the basal body and bilobe structure (BB + Bilobe; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.
    Figure Legend Snippet: De novo assembly of a tagging construct for C-terminal triple-Ty1 tagging using Gibson assembly [A ] Schematic of the plasmid, showing the last 500 bp of the 4400 gene, which functions as a targeting segment ( 1 ; 4400 Cod), the triple-Ty1 tag ( 2; 3X Ty1), the alpha/beta tubulin intergenic region ( 3 ; INTER), the puromycin resistance gene ( 4 ; PAC), a 500 bp segment of the 3′ UTR of 4400 used for targeting ( 5 ; 3′ UTR), and the endogenous tagging vector backbone for inducible expression (ET vector, 6 ). Sizes for each DNA segment are shown in parentheses. [ B ] Agarose gel showing the DNA segments used for Gibson assembly ( 1–6 , as labeled in A ), and the product ( P ) of the assembly digested with PacI and NsiI to show the tagged insert. The asterisk in lane 6 denotes the plasmid backbone of the ET vector, which was used for the Gibson reaction. [ C ] Cells containing the 4400 endogenous tagging construct were fixed and stained with an antibody that detects the basal body and bilobe structure (BB + Bilobe; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.

    Techniques Used: Construct, Plasmid Preparation, Expressing, Agarose Gel Electrophoresis, Labeling, Staining, Fluorescence, Microscopy

    21) Product Images from "A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors"

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.117960

    Novel biosensors from DRIVER exhibit high affinities and selectivities. a. Schematic of overall selection workflow via DRIVER. A high-diversity DNA library of potential RNA biosensors and desired ligands are used as inputs to DRIVER. For each round of selection, the library is transcribed to RNA with or without a ligand mixture present. For rounds with ligands present (yellow) the cDNA corresponding to uncleaved product are amplified with a PCR primer specific to the prefix attached to the input to the current round. For selection rounds without ligand present (blue), the cleaved product is prepended with a new prefix using the regeneration method shown in Fig. 2 and is amplified using a PCR primer specific to this prefix. b. Comparison of cleavage fractions in the presence and absence of the ligand mixture determined via CleaveSeq for a library enriched by DRIVER. Top panel, the enriched library of the S3 selection at round 74 was constricted and CleaveSeq applied in the presence and absence of ligand mixture T1b (N=4,328; at least 30 reads/sequence in each condition). Bottom panel, the same data plotted with the ratio of the cleavage fractions in the presence and absence of the ligand mixture on the x-axis and the standard error of the ratio on the y-axis. Dotted diagonal line, delineates the region where a multiple-hypothesis test would reject the null hypothesis of non-switching, with alpha=1/N. In both panels: dashed line, boundary where the fold change of cleavage is at least 3x; red dots, indicate sequences with strong ( > 3.0), significant (as defined for the diagonal line) switching; green crosses, indicate validated aciclovir biosensors that were first identified from this analysis. c. Binding curves of ligands to select biosensors obtained via SPR analysis. Data points correspond to individual measurements across three experiments. Diamonds mark the equilibrium dissociation constants K D , which are numerically shown in the legend as mean of at least 3 experiments (except for Gard-547, as noted in Methods), and error bars indicate s.e.m. d. Comparison of SPR-derived equilibrium K D and CleaveSeq-derived EC 50 ligand concentrations for select biosensors. K D are shown as mean and error bars in s.e.m of at least 3 independent experiments. CleaveSeq-derived EC 50 and error bars are based on triplicate assays over the ligand and concentrations shown in panel e. e. Fold change of cleavage fractions between the +ligand and −ligand conditions for select biosensors and controls (Dataset S3) under 29 different ligand conditions measured via the CleaveSeq assay. Each column represents a single biosensor sequence (indicated by ID number), grouped by the cognate ligand. Each row represents the ligand concentration (μM) at which the CleaveSeq assay was performed. The color of each cell represents the fold change of cleavage between the −ligand and +ligand conditions. These data viewed as individual gradients for select biosensors to individual ligands are provided in Fig. S6. f. Enrichment of select biosensors. Relative abundance, as determined by NGS sequencing, of select biosensors in the selection libraries during selection rounds immediately prior to their discovery. The legend includes the average enrichment/round and extrapolated round 0 fractions based on an exponential fit to these data.
    Figure Legend Snippet: Novel biosensors from DRIVER exhibit high affinities and selectivities. a. Schematic of overall selection workflow via DRIVER. A high-diversity DNA library of potential RNA biosensors and desired ligands are used as inputs to DRIVER. For each round of selection, the library is transcribed to RNA with or without a ligand mixture present. For rounds with ligands present (yellow) the cDNA corresponding to uncleaved product are amplified with a PCR primer specific to the prefix attached to the input to the current round. For selection rounds without ligand present (blue), the cleaved product is prepended with a new prefix using the regeneration method shown in Fig. 2 and is amplified using a PCR primer specific to this prefix. b. Comparison of cleavage fractions in the presence and absence of the ligand mixture determined via CleaveSeq for a library enriched by DRIVER. Top panel, the enriched library of the S3 selection at round 74 was constricted and CleaveSeq applied in the presence and absence of ligand mixture T1b (N=4,328; at least 30 reads/sequence in each condition). Bottom panel, the same data plotted with the ratio of the cleavage fractions in the presence and absence of the ligand mixture on the x-axis and the standard error of the ratio on the y-axis. Dotted diagonal line, delineates the region where a multiple-hypothesis test would reject the null hypothesis of non-switching, with alpha=1/N. In both panels: dashed line, boundary where the fold change of cleavage is at least 3x; red dots, indicate sequences with strong ( > 3.0), significant (as defined for the diagonal line) switching; green crosses, indicate validated aciclovir biosensors that were first identified from this analysis. c. Binding curves of ligands to select biosensors obtained via SPR analysis. Data points correspond to individual measurements across three experiments. Diamonds mark the equilibrium dissociation constants K D , which are numerically shown in the legend as mean of at least 3 experiments (except for Gard-547, as noted in Methods), and error bars indicate s.e.m. d. Comparison of SPR-derived equilibrium K D and CleaveSeq-derived EC 50 ligand concentrations for select biosensors. K D are shown as mean and error bars in s.e.m of at least 3 independent experiments. CleaveSeq-derived EC 50 and error bars are based on triplicate assays over the ligand and concentrations shown in panel e. e. Fold change of cleavage fractions between the +ligand and −ligand conditions for select biosensors and controls (Dataset S3) under 29 different ligand conditions measured via the CleaveSeq assay. Each column represents a single biosensor sequence (indicated by ID number), grouped by the cognate ligand. Each row represents the ligand concentration (μM) at which the CleaveSeq assay was performed. The color of each cell represents the fold change of cleavage between the −ligand and +ligand conditions. These data viewed as individual gradients for select biosensors to individual ligands are provided in Fig. S6. f. Enrichment of select biosensors. Relative abundance, as determined by NGS sequencing, of select biosensors in the selection libraries during selection rounds immediately prior to their discovery. The legend includes the average enrichment/round and extrapolated round 0 fractions based on an exponential fit to these data.

    Techniques Used: Selection, Amplification, Polymerase Chain Reaction, Sequencing, Binding Assay, SPR Assay, Derivative Assay, Concentration Assay, Next-Generation Sequencing

    D e novo R apid I n V itro E volution of R NA biosensors (DRIVER) is a scalable platform that allows automated, parallelized selection of novel biosensors to diverse ligands. a(i) . Conventional selection starts with an RNA library with randomized regions in an unstructured context and chemical conjugation of a single ligand to a solid phase support such as columns or beads. a(ii) . Scalable DRIVER selection begins with an RNA biosensor library with randomized loop regions in a self-cleaving ribozyme framework and conjugation-free ligands as complex mixtures in solution. b(i) . Conventional SELEX (systematic evolution of ligands by exponential enrichment) involves manually iterating through 1. library incubation with a single immobilized ligand, 2. washing away unbound sequences, 3. eluting enriched sequences, 4. regenerating the DNA library template and transcribing the RNA library. b(ii) . DRIVER selection is fully automated with liquid handling, alternately enriching for sequences that co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in the presence of ligands. Liquid handling enables selection against complex mixtures in parallel, continuously generating hits across different rounds. c(i) . An enriched library from conventional SELEX can be transformed into bacteria to isolate sequences from single colonies, or deeply sequenced with next generation sequencing (NGS) to identify enriched sequences. c(ii) . DRIVER evolved libraries are screened using the fully automated CleaveSeq assay, which uses NGS to identify sequences that are cleaved and uncleaved in the absence and presence of ligand, respectively. d(i) . Hits from conventional SELEX require further orthogonal assays to verify ligand-specific binding versus PCR amplicons or non-specific binders to the column or bead. Once verified, extensive optimization is usually required to convert ligand-specific aptamers into biosensors, with no guarantee of success. d(ii) . DRIVER hits are directly functional in live cells, requiring no further optimization. Time on individual panels indicates an estimated duration for each set of experimental procedures, and ∞ indicates that the desired outcome is not achieved.
    Figure Legend Snippet: D e novo R apid I n V itro E volution of R NA biosensors (DRIVER) is a scalable platform that allows automated, parallelized selection of novel biosensors to diverse ligands. a(i) . Conventional selection starts with an RNA library with randomized regions in an unstructured context and chemical conjugation of a single ligand to a solid phase support such as columns or beads. a(ii) . Scalable DRIVER selection begins with an RNA biosensor library with randomized loop regions in a self-cleaving ribozyme framework and conjugation-free ligands as complex mixtures in solution. b(i) . Conventional SELEX (systematic evolution of ligands by exponential enrichment) involves manually iterating through 1. library incubation with a single immobilized ligand, 2. washing away unbound sequences, 3. eluting enriched sequences, 4. regenerating the DNA library template and transcribing the RNA library. b(ii) . DRIVER selection is fully automated with liquid handling, alternately enriching for sequences that co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in the presence of ligands. Liquid handling enables selection against complex mixtures in parallel, continuously generating hits across different rounds. c(i) . An enriched library from conventional SELEX can be transformed into bacteria to isolate sequences from single colonies, or deeply sequenced with next generation sequencing (NGS) to identify enriched sequences. c(ii) . DRIVER evolved libraries are screened using the fully automated CleaveSeq assay, which uses NGS to identify sequences that are cleaved and uncleaved in the absence and presence of ligand, respectively. d(i) . Hits from conventional SELEX require further orthogonal assays to verify ligand-specific binding versus PCR amplicons or non-specific binders to the column or bead. Once verified, extensive optimization is usually required to convert ligand-specific aptamers into biosensors, with no guarantee of success. d(ii) . DRIVER hits are directly functional in live cells, requiring no further optimization. Time on individual panels indicates an estimated duration for each set of experimental procedures, and ∞ indicates that the desired outcome is not achieved.

    Techniques Used: Selection, Conjugation Assay, Incubation, Transformation Assay, Next-Generation Sequencing, Binding Assay, Polymerase Chain Reaction, Functional Assay

    Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p
    Figure Legend Snippet: Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p

    Techniques Used: Selection, Next-Generation Sequencing, In Vivo, Activity Assay, Sequencing, Derivative Assay, Ligation, Polymerase Chain Reaction, Amplification

    22) Product Images from "Toll-like receptor dual-acting agonists are potent inducers of PBMC-produced cytokines that inhibit hepatitis B virus production in primary human hepatocytes"

    Article Title: Toll-like receptor dual-acting agonists are potent inducers of PBMC-produced cytokines that inhibit hepatitis B virus production in primary human hepatocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69614-7

    Reduction of total HBV DNA ( A ) but not cccDNA ( B ) in HBV-infected PHH by CM from TLR agonist-stimulated PBMCs. PHH were infected with HBV and cultured for 3 days followed by addition of CpG-A-CM, GS-9620[L]-CM, GS-9620[H]-CM or R848–CM (diluted 1:10) or 1,000 IU of IFN-α or IFN-λ. CM was added again 6 DPI. Cells were grown for 3 more days and the quantities of total HBV DNA and cccDNA were determined by qPCR. Data are shown as mean ± SEM with PHH from three donors (N = 3) for CpG-A-CM, GS-9620[L]-CM, and with PHH from one donor (N = 1) for GS-9620[H]-CM, R848-CM (two biological replicates) * p
    Figure Legend Snippet: Reduction of total HBV DNA ( A ) but not cccDNA ( B ) in HBV-infected PHH by CM from TLR agonist-stimulated PBMCs. PHH were infected with HBV and cultured for 3 days followed by addition of CpG-A-CM, GS-9620[L]-CM, GS-9620[H]-CM or R848–CM (diluted 1:10) or 1,000 IU of IFN-α or IFN-λ. CM was added again 6 DPI. Cells were grown for 3 more days and the quantities of total HBV DNA and cccDNA were determined by qPCR. Data are shown as mean ± SEM with PHH from three donors (N = 3) for CpG-A-CM, GS-9620[L]-CM, and with PHH from one donor (N = 1) for GS-9620[H]-CM, R848-CM (two biological replicates) * p

    Techniques Used: Infection, Cell Culture, Real-time Polymerase Chain Reaction

    23) Product Images from "The Clinical Influence of Autophagy-Associated Proteins on Human Lung Cancer"

    Article Title: The Clinical Influence of Autophagy-Associated Proteins on Human Lung Cancer

    Journal: Disease Markers

    doi: 10.1155/2018/8314963

    ), since no PCR products were observed most probably due to degraded genomic DNA. U: unmethylated; M: methylated.
    Figure Legend Snippet: ), since no PCR products were observed most probably due to degraded genomic DNA. U: unmethylated; M: methylated.

    Techniques Used: Polymerase Chain Reaction, Methylation

    24) Product Images from "Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells"

    Article Title: Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells

    Journal: Nature Communications

    doi: 10.1038/ncomms14686

    Expression of ERBB2 and oncogenic KRAS along with tumour suppressor inactivation immortalizes purified primary human duct cells. ( a ) Schematic of lentiviral constructs encoding H2B-mCherry and human ERBB2 . ( b ) Genomic DNA PCR confirming the presence of lentiviral transgenes in uninfected (S2 and S3) and infected (S2 KECST and S3 KECST) spheres. ( c ) Relative mRNA expression level of oncogenic KRAS and ERBB2 transgenes in S2 KECST (left) and S3 KECST (right) spheres; n =2. Error bars=s.d. ( d ) Indel efficiency of each indicated genomic locus assessed by TIDE analysis. ( e ) Quantification of the total cell number in each cell passage of CD133 + cells infected with indicated combinations of lentiviruses. ( f ) Representative stereoscopic and H E staining images of KECST spheres after 85 days in culture. Scale bars, 200 μm.
    Figure Legend Snippet: Expression of ERBB2 and oncogenic KRAS along with tumour suppressor inactivation immortalizes purified primary human duct cells. ( a ) Schematic of lentiviral constructs encoding H2B-mCherry and human ERBB2 . ( b ) Genomic DNA PCR confirming the presence of lentiviral transgenes in uninfected (S2 and S3) and infected (S2 KECST and S3 KECST) spheres. ( c ) Relative mRNA expression level of oncogenic KRAS and ERBB2 transgenes in S2 KECST (left) and S3 KECST (right) spheres; n =2. Error bars=s.d. ( d ) Indel efficiency of each indicated genomic locus assessed by TIDE analysis. ( e ) Quantification of the total cell number in each cell passage of CD133 + cells infected with indicated combinations of lentiviruses. ( f ) Representative stereoscopic and H E staining images of KECST spheres after 85 days in culture. Scale bars, 200 μm.

    Techniques Used: Expressing, Purification, Construct, Polymerase Chain Reaction, Infection, Staining

    Clones with defined genomic mutations form PanIN-like lesions but not PDA. ( a ) Schematic of the sphere clone isolation procedure. See Methods for details. ( b ) Genomic DNA PCR confirming the presence of lentiviral transgenes in clone 3 (left) and clone 4 (right). ( c ) Relative mRNA expression level of oncogenic KRAS transgene (bottom) and the transgene plus endogenous KRAS (top) of clone 3 (left) and clone 4 (right). Error bars=s.d.; n =2. ( d , e ) H E (left) and Alcian blue (right) staining of the transplanted pancreas ID 199 with clone 3 in d and ID 207 with clone 4 in e . Yellow arrows indicate representative abnormal nuclei, red and green arrows indicate necrotic cells and epithelial cell clusters found in the lumen, features of human PanIN2 and 3. Scale bars, 200 μm.
    Figure Legend Snippet: Clones with defined genomic mutations form PanIN-like lesions but not PDA. ( a ) Schematic of the sphere clone isolation procedure. See Methods for details. ( b ) Genomic DNA PCR confirming the presence of lentiviral transgenes in clone 3 (left) and clone 4 (right). ( c ) Relative mRNA expression level of oncogenic KRAS transgene (bottom) and the transgene plus endogenous KRAS (top) of clone 3 (left) and clone 4 (right). Error bars=s.d.; n =2. ( d , e ) H E (left) and Alcian blue (right) staining of the transplanted pancreas ID 199 with clone 3 in d and ID 207 with clone 4 in e . Yellow arrows indicate representative abnormal nuclei, red and green arrows indicate necrotic cells and epithelial cell clusters found in the lumen, features of human PanIN2 and 3. Scale bars, 200 μm.

    Techniques Used: Clone Assay, Isolation, Polymerase Chain Reaction, Expressing, Staining

    Genetic modification of human ductal cell line HPDE induces invasive PDA development. ( a ) Genomic DNA PCR for assessing the presence of lentiviral transgenes in HPDE cells. ( b ) Relative mRNA expression level of oncogenic KRAS and ERBB2 transgene. Error bars=s.d.; n =2. ( c ) Indel efficiency of each indicated genomic locus assessed by TIDE analysis. ( d ) Stereoscopic and representative haematoxylin and eosin (H E) and anti-CK19 (green) immunostaining images of the tumours formed in the transplanted pancreas with HPDE KECST . Red arrows indicate tumour nodules. ( e ) Representative H E staining of lung with metastatic cells found in ID 211. The metastatic cells are CK19 + (green, bottom). ( f ) Stereoscopic and representative H E staining images of the tumours formed in the transplanted pancreas with HPDE KCST . Scale bars, 200 μm.
    Figure Legend Snippet: Genetic modification of human ductal cell line HPDE induces invasive PDA development. ( a ) Genomic DNA PCR for assessing the presence of lentiviral transgenes in HPDE cells. ( b ) Relative mRNA expression level of oncogenic KRAS and ERBB2 transgene. Error bars=s.d.; n =2. ( c ) Indel efficiency of each indicated genomic locus assessed by TIDE analysis. ( d ) Stereoscopic and representative haematoxylin and eosin (H E) and anti-CK19 (green) immunostaining images of the tumours formed in the transplanted pancreas with HPDE KECST . Red arrows indicate tumour nodules. ( e ) Representative H E staining of lung with metastatic cells found in ID 211. The metastatic cells are CK19 + (green, bottom). ( f ) Stereoscopic and representative H E staining images of the tumours formed in the transplanted pancreas with HPDE KCST . Scale bars, 200 μm.

    Techniques Used: Modification, Polymerase Chain Reaction, Expressing, Immunostaining, Staining

    Oncogenic KRAS expression and tumour suppressor inactivation immortalizes purified primary human duct cells. ( a ) Schematic diagram summarizing experimental procedures. ( b ) FACS histogram of the dissociated human adult pancreas stained with antibody specific for CD133. Results are representative of three independent experiments. ( c ) Schematics of lentiviral constructs used and sgRNA sequences for the construction of lentiCRISPRv2. ( d ) Representative images of spheres cultured for 27 days from CD133 + ductal cells infected with combinations of lentiviruses (CTRL- NeoR , Control- NeoR alone; KRAS- NeoR , KRAS- NeoR alone; CTRLmix, Control- NeoR and lentiCRISPRv2-Control; KCST, KRAS- NeoR plus lentiCRISPRv2 against CDKN2A#1, SMAD4#1 and TP53#2). ( e ) Genomic DNA PCR for confirming the presence of lentiviral transgenes in uninfected (S1; Supplementary Table 1 ) and infected (S1 KCST) spheres. bp=base pair. ( f ) Relative mRNA expression level of oncogenic KRAS transgene (left) and the transgene plus endogenous KRAS (right); n =2. ( g ) Indel efficiency of each indicated genomic locus assessed by TIDE analysis, ( h ) Quantification of the total cell number in each cell passage of CD133 + cells infected with indicated combinations of lentiviruses. Data are presented as fold increase over day 1. ( i ) Representative stereoscopic and haematoxylin and eosin (H E) staining images of S1 KCST spheres after 52 days in culture. Error bars=s.d., scale bars, 200 μm.
    Figure Legend Snippet: Oncogenic KRAS expression and tumour suppressor inactivation immortalizes purified primary human duct cells. ( a ) Schematic diagram summarizing experimental procedures. ( b ) FACS histogram of the dissociated human adult pancreas stained with antibody specific for CD133. Results are representative of three independent experiments. ( c ) Schematics of lentiviral constructs used and sgRNA sequences for the construction of lentiCRISPRv2. ( d ) Representative images of spheres cultured for 27 days from CD133 + ductal cells infected with combinations of lentiviruses (CTRL- NeoR , Control- NeoR alone; KRAS- NeoR , KRAS- NeoR alone; CTRLmix, Control- NeoR and lentiCRISPRv2-Control; KCST, KRAS- NeoR plus lentiCRISPRv2 against CDKN2A#1, SMAD4#1 and TP53#2). ( e ) Genomic DNA PCR for confirming the presence of lentiviral transgenes in uninfected (S1; Supplementary Table 1 ) and infected (S1 KCST) spheres. bp=base pair. ( f ) Relative mRNA expression level of oncogenic KRAS transgene (left) and the transgene plus endogenous KRAS (right); n =2. ( g ) Indel efficiency of each indicated genomic locus assessed by TIDE analysis, ( h ) Quantification of the total cell number in each cell passage of CD133 + cells infected with indicated combinations of lentiviruses. Data are presented as fold increase over day 1. ( i ) Representative stereoscopic and haematoxylin and eosin (H E) staining images of S1 KCST spheres after 52 days in culture. Error bars=s.d., scale bars, 200 μm.

    Techniques Used: Expressing, Purification, FACS, Staining, Construct, Cell Culture, Infection, Polymerase Chain Reaction

    25) Product Images from "Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis"

    Article Title: Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1263-4

    NGS-TILLING process. A : A long-term resource for many TILLING screens consisting of a genomic DNA sample and a corresponding cryopreserved sperm sample was prepared from 9,024 F1 ENU-mutagenized male zebrafish. B : Library Pooling. Normalized genomic DNA (gDNA) was pooled twice: first, gDNA from 6 fish was pooled together to make 1,504 6-fish pools in 16 96-well plates. These six-fish pools will be used for HRM identification of carrier fish (step F ). Second, groups of 48 6-fish pools were pooled together into 288-fish pools (a total of 32 288-fish pools). C : Target Preparation. gDNA from 288-fish pools was used as a template for PCR amplification of ~250 bp fragments corresponding to exons of genes of interest using gene-specific primers with P5/P7 SEQ tails (green). After normalization, amplicons from each 288-fish pool were combined and used as template for a brief second PCR that added Nextera index sequences (blue) and Illumina P5/P7 sequences (yellow). D : Sequencing: All amplicons from the entire library were combined and sequenced (Illumina MiSeq platform), generating fully overlapping 250 bp paired-end sequences. E : Data Analysis. Sequence analysis using PELE and PoDATA identified rare deleterious variants (occurring in 1/100 to 1/1000 reads) in single 288-fish pools. F : Deconvolution. A fragment centered on a putative variant call was amplified from each of the 48-six-fish pools used to make up the 288-fish pool in which that variant was detected, and was subjected to High Resolution Melt (HRM) Analysis. Then HRM of the six individual fish in the six-fish pool that showed distinct melting kinetics identified the individual carrier. G : Mutant Recovery. Finally, the presence of the variant identified by PELE and PoDATA was confirmed in that fish by Sanger sequencing. F2 heterozygotes were generated by in vitro fertilization of WT eggs with the corresponding cryopreserved sperm sample.
    Figure Legend Snippet: NGS-TILLING process. A : A long-term resource for many TILLING screens consisting of a genomic DNA sample and a corresponding cryopreserved sperm sample was prepared from 9,024 F1 ENU-mutagenized male zebrafish. B : Library Pooling. Normalized genomic DNA (gDNA) was pooled twice: first, gDNA from 6 fish was pooled together to make 1,504 6-fish pools in 16 96-well plates. These six-fish pools will be used for HRM identification of carrier fish (step F ). Second, groups of 48 6-fish pools were pooled together into 288-fish pools (a total of 32 288-fish pools). C : Target Preparation. gDNA from 288-fish pools was used as a template for PCR amplification of ~250 bp fragments corresponding to exons of genes of interest using gene-specific primers with P5/P7 SEQ tails (green). After normalization, amplicons from each 288-fish pool were combined and used as template for a brief second PCR that added Nextera index sequences (blue) and Illumina P5/P7 sequences (yellow). D : Sequencing: All amplicons from the entire library were combined and sequenced (Illumina MiSeq platform), generating fully overlapping 250 bp paired-end sequences. E : Data Analysis. Sequence analysis using PELE and PoDATA identified rare deleterious variants (occurring in 1/100 to 1/1000 reads) in single 288-fish pools. F : Deconvolution. A fragment centered on a putative variant call was amplified from each of the 48-six-fish pools used to make up the 288-fish pool in which that variant was detected, and was subjected to High Resolution Melt (HRM) Analysis. Then HRM of the six individual fish in the six-fish pool that showed distinct melting kinetics identified the individual carrier. G : Mutant Recovery. Finally, the presence of the variant identified by PELE and PoDATA was confirmed in that fish by Sanger sequencing. F2 heterozygotes were generated by in vitro fertilization of WT eggs with the corresponding cryopreserved sperm sample.

    Techniques Used: Next-Generation Sequencing, Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing, Variant Assay, Mutagenesis, Generated, In Vitro

    26) Product Images from "Genome-wide histone modification profiling of inner cell mass and trophectoderm of bovine blastocysts by RAT-ChIP"

    Article Title: Genome-wide histone modification profiling of inner cell mass and trophectoderm of bovine blastocysts by RAT-ChIP

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0225801

    RAT-ChIP enables genome wide histone modification profiling from 100 cells. A Overview of RAT-ChIP method. B Agarose gel electrophoresis of DNA after chromatin treatment with a combination of restriction enzymes (middle lane) and after tagmentation (left lane). C UCSC genome browser custom histone H3K4me3 and H3K27me3 tracks of RAT-ChIP-seq with 100 and 1,000 K562 cells in comparison with ENCODE data in a genomic region centred around IL17C gene. D Clustered global Pearson correlation heatmap (enrichments in 5kb windows) of RAT-ChIP-seq and different published histone H3K4me3 and H3K27me3 datasets in K562 cells.
    Figure Legend Snippet: RAT-ChIP enables genome wide histone modification profiling from 100 cells. A Overview of RAT-ChIP method. B Agarose gel electrophoresis of DNA after chromatin treatment with a combination of restriction enzymes (middle lane) and after tagmentation (left lane). C UCSC genome browser custom histone H3K4me3 and H3K27me3 tracks of RAT-ChIP-seq with 100 and 1,000 K562 cells in comparison with ENCODE data in a genomic region centred around IL17C gene. D Clustered global Pearson correlation heatmap (enrichments in 5kb windows) of RAT-ChIP-seq and different published histone H3K4me3 and H3K27me3 datasets in K562 cells.

    Techniques Used: Chromatin Immunoprecipitation, Genome Wide, Modification, Agarose Gel Electrophoresis

    27) Product Images from "Prevalence of Human Parvovirus B19 in Neurological Patients: Findings from Region of Western Saudi Arabia"

    Article Title: Prevalence of Human Parvovirus B19 in Neurological Patients: Findings from Region of Western Saudi Arabia

    Journal: Current Health Sciences Journal

    doi: 10.12865/CHSJ.46.01.03

    PCR for HPVB19. Lane 1 and 13 show 100bp DNA ladder, Lane 2-3, 5-11 indicating total 9 PCR positive samples with product size of 102bp. Lane 4 and Lane 12 represent positive and negative control respectively
    Figure Legend Snippet: PCR for HPVB19. Lane 1 and 13 show 100bp DNA ladder, Lane 2-3, 5-11 indicating total 9 PCR positive samples with product size of 102bp. Lane 4 and Lane 12 represent positive and negative control respectively

    Techniques Used: Polymerase Chain Reaction, Negative Control

    28) Product Images from "Late-phase immune responses limiting oocyst survival are independent of TEP1 function yet display strain specific differences in Anopheles gambiae"

    Article Title: Late-phase immune responses limiting oocyst survival are independent of TEP1 function yet display strain specific differences in Anopheles gambiae

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2308-0

    Expression of LL3 varies among different molecular forms of An. gambiae . A PCR-RFLP was performed to identify the molecular forms (M and S) that distinguish An . gambiae strains used in this study (X1, TEP1 mutant, G3, and Keele strains) as well as a known M-form strain (Ngousso) of An. coluzzii. DNA was prepared from individual mosquito samples for each strain and displayed consistent RFLP patterns for all mosquitoes analyzed. Individuals from each strain were used to create a representative image of the RFLP patterns for each mosquito strain ( a ). The X1, TEP1 mutant, and G3 strains display a hybrid pattern (M/S) of three bands (367, 257 and 110 bp), while the Keele and Ngousso strains are M molecular form (367 bp). Relative LL3 expression in the Keele strain is higher than those of the X1 and G3 strains at 24 h P . berghei infection ( b ), while there is no difference of STAT-A expression among mosquito strains ( c ). To examine the effects of gene-silencing on hemocyte differentiation, the proportion of granulocytes (out of total cell population) was examined in individual mosquitoes 4 days post-infection with P. berghei . The percentage of granulocytes were measured in dsGFP-, dsLL3-, or dsSTAT-A-treated mosquitoes in both the G3 ( d ) and Keele ( e ) strains. STAT-A- silencing abrogated hemocyte differentiation in both G3 and Keele mosquitoes ( d ) and ( e ), whereas LL3-silencing only influenced the Keele strain ( e ). Gene expression data were analyzed with a one-way ANOVA and Tukey post-hoc test, while granulocyte percentages were evaluated by Kruskal-Wallis with a Dunn’s post-hoc test. Asterisks denote significance (* P
    Figure Legend Snippet: Expression of LL3 varies among different molecular forms of An. gambiae . A PCR-RFLP was performed to identify the molecular forms (M and S) that distinguish An . gambiae strains used in this study (X1, TEP1 mutant, G3, and Keele strains) as well as a known M-form strain (Ngousso) of An. coluzzii. DNA was prepared from individual mosquito samples for each strain and displayed consistent RFLP patterns for all mosquitoes analyzed. Individuals from each strain were used to create a representative image of the RFLP patterns for each mosquito strain ( a ). The X1, TEP1 mutant, and G3 strains display a hybrid pattern (M/S) of three bands (367, 257 and 110 bp), while the Keele and Ngousso strains are M molecular form (367 bp). Relative LL3 expression in the Keele strain is higher than those of the X1 and G3 strains at 24 h P . berghei infection ( b ), while there is no difference of STAT-A expression among mosquito strains ( c ). To examine the effects of gene-silencing on hemocyte differentiation, the proportion of granulocytes (out of total cell population) was examined in individual mosquitoes 4 days post-infection with P. berghei . The percentage of granulocytes were measured in dsGFP-, dsLL3-, or dsSTAT-A-treated mosquitoes in both the G3 ( d ) and Keele ( e ) strains. STAT-A- silencing abrogated hemocyte differentiation in both G3 and Keele mosquitoes ( d ) and ( e ), whereas LL3-silencing only influenced the Keele strain ( e ). Gene expression data were analyzed with a one-way ANOVA and Tukey post-hoc test, while granulocyte percentages were evaluated by Kruskal-Wallis with a Dunn’s post-hoc test. Asterisks denote significance (* P

    Techniques Used: Expressing, Polymerase Chain Reaction, Mutagenesis, Infection

    29) Product Images from "Natural Competence Is Common among Clinical Isolates of Veillonella parvula and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome"

    Article Title: Natural Competence Is Common among Clinical Isolates of Veillonella parvula and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00139

    Allelic replacement mutagenesis of recA and a putative competence gene locus. (A) Chromosome maps of regions targeted for allelic exchange mutagenesis. Open reading frames (ORFs) are all drawn to scale. Red arrows indicate the locations of primers used to amplify homologous fragments used for mutagenesis. For both loci, the region between the two primer sets was replaced with a tetM cassette. NCBI gene locus tags are written in blue with the final two digits of the locus tag listed directly below their respective ORFs. For the competence gene locus map, the bottom row (N methyl 2) indicates whether a putative A24 prepilin cleavage/N-terminal methylation motif is present within the ORF. This motif is commonly found among type IV-like prepilins. (B) V. parvula strain SKV38 was transformed using either SK medium (top row) or THL medium (bottom row) together with PCR products containing allelic exchange mutagenesis constructs for both recA and the competence gene locus (CGL). Ten microliters of the transformation reaction was spotted onto selective medium to isolate transformants. Samples from left to right are: SKV38 + Δ recA PCR, SKV38 (no DNA), SKV38 + ΔCGL PCR, and SKV38 (no DNA). (C) Transformants from both the Δ recA and ΔCGL transformations were tested via PCR to confirm the expected genotypes. Samples from left to right are: 1. wild-type gDNA + recA locus external primers (predicted size 2,846 bp); 2. wild-type gDNA + tetM primers (no predicted amplicon); 3. Δ recA gDNA + recA locus external primers (predicted size 4,176 bp); 4. Δ recA gDNA + recA locus upstream external primer/ tetM reverse primer (predicted size 3,162 bp); 5. Δ recA gDNA + tetM forward primer/ recA locus external downstream primer (predicted size 3,086 bp); 6. wild-type gDNA + CGL external primers (predicted size 7,602 bp); 7. wild-type gDNA + tetM primers (no predicted amplicon); 8. ΔCGL gDNA + CGL external primers (predicted size 3,938 bp); 9. ΔCGL gDNA + CGL upstream external primer/ tetM reverse primer (predicted size 2,992 bp); 10. ΔCGL gDNA + tetM forward primer/CGL external downstream primer (predicted size 3,018 bp). (D) Confirmed deletion mutants of recA and the competence gene locus were transformed with gDNA derived from a spontaneous spectinomycin resistant mutant of strain SKV38. Ten microliters of the transformation reaction and three consecutive serial dilutions were spotted onto selective medium to isolate transformants. Samples from left to right are: wild-type (WT), Δ recA ( recA ), and ΔCGL (CGL).
    Figure Legend Snippet: Allelic replacement mutagenesis of recA and a putative competence gene locus. (A) Chromosome maps of regions targeted for allelic exchange mutagenesis. Open reading frames (ORFs) are all drawn to scale. Red arrows indicate the locations of primers used to amplify homologous fragments used for mutagenesis. For both loci, the region between the two primer sets was replaced with a tetM cassette. NCBI gene locus tags are written in blue with the final two digits of the locus tag listed directly below their respective ORFs. For the competence gene locus map, the bottom row (N methyl 2) indicates whether a putative A24 prepilin cleavage/N-terminal methylation motif is present within the ORF. This motif is commonly found among type IV-like prepilins. (B) V. parvula strain SKV38 was transformed using either SK medium (top row) or THL medium (bottom row) together with PCR products containing allelic exchange mutagenesis constructs for both recA and the competence gene locus (CGL). Ten microliters of the transformation reaction was spotted onto selective medium to isolate transformants. Samples from left to right are: SKV38 + Δ recA PCR, SKV38 (no DNA), SKV38 + ΔCGL PCR, and SKV38 (no DNA). (C) Transformants from both the Δ recA and ΔCGL transformations were tested via PCR to confirm the expected genotypes. Samples from left to right are: 1. wild-type gDNA + recA locus external primers (predicted size 2,846 bp); 2. wild-type gDNA + tetM primers (no predicted amplicon); 3. Δ recA gDNA + recA locus external primers (predicted size 4,176 bp); 4. Δ recA gDNA + recA locus upstream external primer/ tetM reverse primer (predicted size 3,162 bp); 5. Δ recA gDNA + tetM forward primer/ recA locus external downstream primer (predicted size 3,086 bp); 6. wild-type gDNA + CGL external primers (predicted size 7,602 bp); 7. wild-type gDNA + tetM primers (no predicted amplicon); 8. ΔCGL gDNA + CGL external primers (predicted size 3,938 bp); 9. ΔCGL gDNA + CGL upstream external primer/ tetM reverse primer (predicted size 2,992 bp); 10. ΔCGL gDNA + tetM forward primer/CGL external downstream primer (predicted size 3,018 bp). (D) Confirmed deletion mutants of recA and the competence gene locus were transformed with gDNA derived from a spontaneous spectinomycin resistant mutant of strain SKV38. Ten microliters of the transformation reaction and three consecutive serial dilutions were spotted onto selective medium to isolate transformants. Samples from left to right are: wild-type (WT), Δ recA ( recA ), and ΔCGL (CGL).

    Techniques Used: Mutagenesis, Methylation, Transformation Assay, Polymerase Chain Reaction, Construct, Amplification, Derivative Assay

    30) Product Images from "Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale"

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03123-18

    (A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through PCR driven by primer pair malG _F/ malG _R (C+, probe positive control represented by pGEM-T- malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.
    Figure Legend Snippet: (A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through PCR driven by primer pair malG _F/ malG _R (C+, probe positive control represented by pGEM-T- malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.

    Techniques Used: Southern Blot, Infection, Labeling, Amplification, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Electrophoresis

    31) Product Images from "Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis"

    Article Title: Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis

    Journal: Archaea

    doi: 10.1155/2013/185250

    Characterization of the comE ::Tn5 mutant strain S201. (a) Genetic maps of the gene comE in the wild type strain and the comE ::Tn5 mutant strain S201. Numbers indicate the MMP identification, and the black arrows indicate primers used for PCR amplification. (b) Genotypic characterization of the comE ::Tn5 mutant strain 201 by PCR amplification. Lane 1, standard 1 kb ladder (New England Biolab); Lanes 2 and 3, PCR amplifications of comE using primers comEF (Af) and comER (Ar) for genomic DNA of the comE ::Tn5 mutant strain S201 and wild type strain S2, respectively; Lanes 4 and 5, PCR amplifications using a primer from the end of the transposon (KAN-2RP-1out2; Br) and a primer that binds upstream of the gene comE (Bf) for the same DNAs. (c) Growth of the wild type and comE ::Tn5 mutant strain 201 in minimal medium + acetate (McNA) and McNA supplemented with coenzyme M after three passages. Grey, comE ::Tn5 McNA; green, comE ::Tn5 McNA + CoM; red, S2 McNA; blue, S2 McNA + CoM. Representative error bars indicate the standard deviation of three independent cultures. The inoculum was ~1 × 10 4 cells.
    Figure Legend Snippet: Characterization of the comE ::Tn5 mutant strain S201. (a) Genetic maps of the gene comE in the wild type strain and the comE ::Tn5 mutant strain S201. Numbers indicate the MMP identification, and the black arrows indicate primers used for PCR amplification. (b) Genotypic characterization of the comE ::Tn5 mutant strain 201 by PCR amplification. Lane 1, standard 1 kb ladder (New England Biolab); Lanes 2 and 3, PCR amplifications of comE using primers comEF (Af) and comER (Ar) for genomic DNA of the comE ::Tn5 mutant strain S201 and wild type strain S2, respectively; Lanes 4 and 5, PCR amplifications using a primer from the end of the transposon (KAN-2RP-1out2; Br) and a primer that binds upstream of the gene comE (Bf) for the same DNAs. (c) Growth of the wild type and comE ::Tn5 mutant strain 201 in minimal medium + acetate (McNA) and McNA supplemented with coenzyme M after three passages. Grey, comE ::Tn5 McNA; green, comE ::Tn5 McNA + CoM; red, S2 McNA; blue, S2 McNA + CoM. Representative error bars indicate the standard deviation of three independent cultures. The inoculum was ~1 × 10 4 cells.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Standard Deviation

    32) Product Images from "EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA"

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884692

    EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.
    Figure Legend Snippet: EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.

    Techniques Used: Lambda DNA Preparation, Methylation, Sequencing, Polymerase Chain Reaction

    33) Product Images from "Novel Diversity and Virulence Patterns Found in New Isolates of Cydia pomonella Granulovirus from China"

    Article Title: Novel Diversity and Virulence Patterns Found in New Isolates of Cydia pomonella Granulovirus from China

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02000-19

    Agarose gel of PCR fragments of 2 × 12-bp-repeat insertion region of the pe38 gene from isolates CpGV-ZY, -JQ, -ALE, -KS1, -ZY2, -WW, -M, -S, and -E2. CK, negative control; SM, size marker (GeneRuler 1 kb Plus DNA Ladder; Thermo Scientific, Carlsbad, CA, USA). Sizes of bands are given in bp.
    Figure Legend Snippet: Agarose gel of PCR fragments of 2 × 12-bp-repeat insertion region of the pe38 gene from isolates CpGV-ZY, -JQ, -ALE, -KS1, -ZY2, -WW, -M, -S, and -E2. CK, negative control; SM, size marker (GeneRuler 1 kb Plus DNA Ladder; Thermo Scientific, Carlsbad, CA, USA). Sizes of bands are given in bp.

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

    34) Product Images from "Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model, et al. Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model"

    Article Title: Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model, et al. Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model

    Journal: Cancer Science

    doi: 10.1111/cas.13880

    Suppression of aberrant DNA methylation induction by treatment with antibiotics. A, DNA methylation levels were analyzed by quantitative methylation‐specific PCR ( qMSP ) of four marker genes in colon tumors. High levels of methylation were observed. B, DNA methylation levels at 10 weeks after treatment with azoxymethane ( AOM )/antibiotics were analyzed by qMSP of four marker genes in colonic epithelial cells. Giving AOM /dextran sulfate sodium ( DSS ) induced aberrant methylation in three markers, which was suppressed by treatment with antibiotics. Horizontal bars indicate the average. * P
    Figure Legend Snippet: Suppression of aberrant DNA methylation induction by treatment with antibiotics. A, DNA methylation levels were analyzed by quantitative methylation‐specific PCR ( qMSP ) of four marker genes in colon tumors. High levels of methylation were observed. B, DNA methylation levels at 10 weeks after treatment with azoxymethane ( AOM )/antibiotics were analyzed by qMSP of four marker genes in colonic epithelial cells. Giving AOM /dextran sulfate sodium ( DSS ) induced aberrant methylation in three markers, which was suppressed by treatment with antibiotics. Horizontal bars indicate the average. * P

    Techniques Used: DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Marker

    35) Product Images from "Detection of class 1 integron-associated gene cassettes and tetracycline resistance genes in Escherichia coli isolated from ready to eat vegetables"

    Article Title: Detection of class 1 integron-associated gene cassettes and tetracycline resistance genes in Escherichia coli isolated from ready to eat vegetables

    Journal: Annals of Medicine and Surgery

    doi: 10.1016/j.amsu.2020.04.044

    Characterisation of Antibiotic Resistance Gene Cassettes in Class 1 Integrons. All TRE isolates contain class I integrons, shown by the PCR product bands at 483 bp (A); and contain the tetA gene shown by the PCR product at 210 bp (B). M is DNA marker; number of isolate was marked by number 1-18.
    Figure Legend Snippet: Characterisation of Antibiotic Resistance Gene Cassettes in Class 1 Integrons. All TRE isolates contain class I integrons, shown by the PCR product bands at 483 bp (A); and contain the tetA gene shown by the PCR product at 210 bp (B). M is DNA marker; number of isolate was marked by number 1-18.

    Techniques Used: Polymerase Chain Reaction, Marker

    36) Product Images from "Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology"

    Article Title: Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology

    Journal: Molecules

    doi: 10.3390/molecules23123178

    Enzyme-assisted activation and deactivation of fluorescent responses. ( a ) Co-transcriptional assembly of fluorets in the presence of DFHBI-1T. ( b ) DNase-assisted production of active fluorets from RNA/DNA duplexes, and their further deactivation with RNases.
    Figure Legend Snippet: Enzyme-assisted activation and deactivation of fluorescent responses. ( a ) Co-transcriptional assembly of fluorets in the presence of DFHBI-1T. ( b ) DNase-assisted production of active fluorets from RNA/DNA duplexes, and their further deactivation with RNases.

    Techniques Used: Activation Assay

    37) Product Images from "Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors"

    Article Title: Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors

    Journal: Cell Adhesion & Migration

    doi: 10.4161/cam.5.5.17949

    Activation of ITGA9 expression in MCF7 breast carcinoma cells by 5-deoxyazacytidine treatment. (A) Representative gel showing ITGA9 amplified by multiplex RT-PCR. (B) Intensity of the amplified ITGA9 DNA fragments normalized to that of GAPDH (TotalLab
    Figure Legend Snippet: Activation of ITGA9 expression in MCF7 breast carcinoma cells by 5-deoxyazacytidine treatment. (A) Representative gel showing ITGA9 amplified by multiplex RT-PCR. (B) Intensity of the amplified ITGA9 DNA fragments normalized to that of GAPDH (TotalLab

    Techniques Used: Activation Assay, Expressing, Amplification, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

    Multiplex RT-PCR analysis of ITGA9 expression with different primer pairs. (A) Scheme of the primers used in the study. (B) Representative gel from multiplex RT-PCR. DNA fragments amplified with two different primers pairs are shown. GAPDH expression
    Figure Legend Snippet: Multiplex RT-PCR analysis of ITGA9 expression with different primer pairs. (A) Scheme of the primers used in the study. (B) Representative gel from multiplex RT-PCR. DNA fragments amplified with two different primers pairs are shown. GAPDH expression

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

    38) Product Images from "Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model"

    Article Title: Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00006

    Representative bioanalyzer images of freshly isolated and bead-purified cfDNA. (A–C) Healthy human control, (D–F) WT mouse, and (G–I) PDAC mouse. The left columns depict the freshly isolated starting cfDNA (green arrow and dotted line) and the contaminating high molecular weight DNA (black arrow) evident by a wide peak. The middle column depicts the high molecular weight DNA removed from the same samples shown in (A–G) by the SPRI AMPure bead purification step after the 0.5X dilution step. The right column shows the purified cfDNA as recovered form the SPRI AMPure bead purification step after the 1.6X dilution step from the same samples shown in (A–G) . The desired cfDNA peak is visible ∼150–200 bp (green arrow and dotted line).
    Figure Legend Snippet: Representative bioanalyzer images of freshly isolated and bead-purified cfDNA. (A–C) Healthy human control, (D–F) WT mouse, and (G–I) PDAC mouse. The left columns depict the freshly isolated starting cfDNA (green arrow and dotted line) and the contaminating high molecular weight DNA (black arrow) evident by a wide peak. The middle column depicts the high molecular weight DNA removed from the same samples shown in (A–G) by the SPRI AMPure bead purification step after the 0.5X dilution step. The right column shows the purified cfDNA as recovered form the SPRI AMPure bead purification step after the 1.6X dilution step from the same samples shown in (A–G) . The desired cfDNA peak is visible ∼150–200 bp (green arrow and dotted line).

    Techniques Used: Isolation, Purification, Molecular Weight

    39) Product Images from "The New Immortalized Uroepithelial Cell Line HBLAK Contains Defined Genetic Aberrations Typical of Early Stage Urothelial Tumors"

    Article Title: The New Immortalized Uroepithelial Cell Line HBLAK Contains Defined Genetic Aberrations Typical of Early Stage Urothelial Tumors

    Journal: Bladder Cancer (Amsterdam, Netherlands)

    doi: 10.3233/BLC-160065

    Characterization of genetic changes in HBLAK cells. (a) Deletion of CDKN2A / p16 INK 4 exon 3 confirmed by PCR amplification of genomic DNA of various HBLAK passages. Urothelial carcinoma cell lines retaining expression of p16 INK 4 (VM-CUB1, 639-V) and experimentally immortalized normal NHUC cells (TERT-NHUC) served as positive controls. The UC cell line RT-112 with a homozygous deletion of CDKN2A served as a negative control. (b) Sanger sequencing revealed the most common TERT promoter mutation (C228T) in HBLAK, whereas C250 was retained. (c) TERT mRNA expression was determined by real time qRT-PCR in different passages of HBLAK cells compared to non-immortalized NHUC cells of different patients, TERT-NHUC and further cell lines with wildtype (HEK293, 253J) and mutant (VM-CUB1) TERT promoter. TBP was used as a reference gene. Note that TERT expression in TERT-NHUC is much higher than that of all other cell lines (76.0). (d) The full-length transcript of p53 was detectable in HBLAK by RT-PCR, but weakly expressed compared to urothelial carcinoma cell lines J82 and VM-CUB1. The amplification product was used for subsequent Sanger sequencing of the amplicon revealing that HBLAK cells contain wildtype p53 . Left lane: size markers. (e) Overview of variant frequency by chromosome in HBLAK cells compared to the hg19 reference genome per chromosome as detected by exome sequencing. For each chromosome, frameshift (blue), nonframeshift (red), and nonsynonymous (green) variants are indicated. Other types are detailed in Table 4 and all variants are listed individually in supplementary file, sheet 2 .
    Figure Legend Snippet: Characterization of genetic changes in HBLAK cells. (a) Deletion of CDKN2A / p16 INK 4 exon 3 confirmed by PCR amplification of genomic DNA of various HBLAK passages. Urothelial carcinoma cell lines retaining expression of p16 INK 4 (VM-CUB1, 639-V) and experimentally immortalized normal NHUC cells (TERT-NHUC) served as positive controls. The UC cell line RT-112 with a homozygous deletion of CDKN2A served as a negative control. (b) Sanger sequencing revealed the most common TERT promoter mutation (C228T) in HBLAK, whereas C250 was retained. (c) TERT mRNA expression was determined by real time qRT-PCR in different passages of HBLAK cells compared to non-immortalized NHUC cells of different patients, TERT-NHUC and further cell lines with wildtype (HEK293, 253J) and mutant (VM-CUB1) TERT promoter. TBP was used as a reference gene. Note that TERT expression in TERT-NHUC is much higher than that of all other cell lines (76.0). (d) The full-length transcript of p53 was detectable in HBLAK by RT-PCR, but weakly expressed compared to urothelial carcinoma cell lines J82 and VM-CUB1. The amplification product was used for subsequent Sanger sequencing of the amplicon revealing that HBLAK cells contain wildtype p53 . Left lane: size markers. (e) Overview of variant frequency by chromosome in HBLAK cells compared to the hg19 reference genome per chromosome as detected by exome sequencing. For each chromosome, frameshift (blue), nonframeshift (red), and nonsynonymous (green) variants are indicated. Other types are detailed in Table 4 and all variants are listed individually in supplementary file, sheet 2 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Expressing, Negative Control, Sequencing, Mutagenesis, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Variant Assay

    40) Product Images from "Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria"

    Article Title: Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

    Journal: mSystems

    doi: 10.1128/mSystems.00143-17

    Golden Gate assembly of transposon delivery vectors from part vectors. (A to E) The five part vectors that are used for Golden Gate assembly of the magic pool transposon delivery vectors: part1 vector, part2 vector, part3 vector, barcoded part4 vector, and part5 vector (not drawn to scale). We show the sequences of the 4-nucleotide overhangs for Golden Gate assembly. (F) The transposon delivery vector with DNA barcode. ColE1 is the replication origin ColE1. cat is the chloramphenicol resistance gene. GFP is green fluorescent protein. oriT is the origin of transfer. AmpR is the beta-lactam resistance gene. R6K is a conditional replication origin. N20 is random 20-nucleotide DNA barcode.
    Figure Legend Snippet: Golden Gate assembly of transposon delivery vectors from part vectors. (A to E) The five part vectors that are used for Golden Gate assembly of the magic pool transposon delivery vectors: part1 vector, part2 vector, part3 vector, barcoded part4 vector, and part5 vector (not drawn to scale). We show the sequences of the 4-nucleotide overhangs for Golden Gate assembly. (F) The transposon delivery vector with DNA barcode. ColE1 is the replication origin ColE1. cat is the chloramphenicol resistance gene. GFP is green fluorescent protein. oriT is the origin of transfer. AmpR is the beta-lactam resistance gene. R6K is a conditional replication origin. N20 is random 20-nucleotide DNA barcode.

    Techniques Used: Plasmid Preparation

    Overview of the magic pool strategy. (A) Basic structure of a typical transposon delivery vector (not drawn to scale). The inverted repeat (IR) for the specific transposase is indicated. We dissected the transposon delivery vector into five different parts compatible with Golden Gate assembly, and the different parts are indicated by different colors. (B) General workflow of construction and application of magic pools. In step 1, variants of the five different parts are designed, cloned into a part-holding vector, confirmed by sequencing, and archived. In step 2, the part vectors are mixed and assembled using Golden Gate assembly to produce the magic pools of transposon delivery vectors. In step 3, the magic pool vectors are characterized by DNA sequencing whereby each unique DNA barcode (random 20-nucleotide DNA barcode [N20]) is linked to a specific combination of parts. In step 4, preliminary mutant libraries of approximately 5,000 CFU are made using the magic pool, and TnSeq is performed to link the DNA barcode to the insertion site, thereby simultaneously assessing the efficacy of the vectors in the magic pool. ID, identification. In step 5, an effective vector is reassembled using the archived parts, fully barcoded with millions of random DNA barcodes, and a full RB-TnSeq transposon mutant library is constructed. oriT is the origin of transfer. AmpR is the beta-lactam resistance cassette. R6K is the conditional replication origin.
    Figure Legend Snippet: Overview of the magic pool strategy. (A) Basic structure of a typical transposon delivery vector (not drawn to scale). The inverted repeat (IR) for the specific transposase is indicated. We dissected the transposon delivery vector into five different parts compatible with Golden Gate assembly, and the different parts are indicated by different colors. (B) General workflow of construction and application of magic pools. In step 1, variants of the five different parts are designed, cloned into a part-holding vector, confirmed by sequencing, and archived. In step 2, the part vectors are mixed and assembled using Golden Gate assembly to produce the magic pools of transposon delivery vectors. In step 3, the magic pool vectors are characterized by DNA sequencing whereby each unique DNA barcode (random 20-nucleotide DNA barcode [N20]) is linked to a specific combination of parts. In step 4, preliminary mutant libraries of approximately 5,000 CFU are made using the magic pool, and TnSeq is performed to link the DNA barcode to the insertion site, thereby simultaneously assessing the efficacy of the vectors in the magic pool. ID, identification. In step 5, an effective vector is reassembled using the archived parts, fully barcoded with millions of random DNA barcodes, and a full RB-TnSeq transposon mutant library is constructed. oriT is the origin of transfer. AmpR is the beta-lactam resistance cassette. R6K is the conditional replication origin.

    Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, DNA Sequencing, Mutagenesis, Construct

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    Amplification:

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    Mutagenesis:

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    Size-exclusion Chromatography:

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