genomic dna Thermo Fisher Search Results


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  • 99
    Thermo Fisher genomic dna gdna
    CRISPR/Cas9-mediated editing of the CFTR gene reduces the expression of CFTR. Ai : T7 endonuclease I (T7EI)-digested heteroduplexed PCR products, which contained indels (insertions/deletions) near the predicted lengths (228 and 168 bp), that were amplified from genomic <t>DNA</t> extracted from single amacrine cells transfected with pCRISPR-CFTR and pSpCas9. T7EI only digested heteroduplexed PCR products amplified from the amacrine cell transfected with pCRISPR-CFTR. Aii : T7EI-digested heteroduplexed PCR products, containing indels near the predicted lengths (228 and 168 bp), that were amplified from genomic DNA extracted from a population of cultured retinal cells transfected with pCRISPR-CFTRtd. T7EI did not digest heteroduplexed PCR products amplified from genomic DNA extracted from a population of cells transfected with pSpCas9td. B–G : representative amacrine cells transfected with pSpCas9td ( B–D ) and pCRISPR-CFTRtd ( E–G ). Anti-CFTR labeling ( C and F ) and merged images ( D and G ) are shown. White boxes in B–G are shown at higher magnification in Bi–Gi , respectively. Scale bars are 10 µm ( B–G ) and 5 µm ( Bi–Gi ). H–K : data from analyzed processes from cells transfected with the pSPCas9td construct and processes from cells transfected with pCRISPR-CFTRtd. Shown are mean total CFTR area ( H ), the mean CFTR object size ( I ), the mean CFTR intensity ( J ), and the mean tdTomato intensity within the CFTR object domains ( K ); n = 10 for both groups; A.U., arbitrary units. ** P
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    Thermo Fisher easy dna genomic dna gdna purification kit
    Comparison of <t>DNA</t> extraction methods. (A) Experimental design. Human feces, pig feces, and hospital sewage were extracted using seven different DNA extraction methods ( Table 1 ): InnuPure C16, MagNA Pure LC DNA isolation kit III, Easy-DNA <t>gDNA</t> purification kit, MP FastDNA Spin kit, PowerSoil DNA isolation kit, QIAamp DNA stool minikit, and QIAamp DNA stool minikit plus bead beating (for details, see Materials and Methods). DNA concentration, purity, and stability were examined, and microbial community composition was determined using 16S rRNA gene profiling and metagenomics (selected samples). (B) DNA from each method was dissolved in 100 µl solution, and DNA concentrations were determined using Qubit dsDNA BR assay kit measurements. Values represent averages from duplicate or triplicate DNA extractions (see also Table S1A in the supplemental material). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (see also Table S1B ).
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    99
    Thermo Fisher genomic dna
    Gene trap of Gldc ablates GCS activity. The GCS, comprising GLDC, GCSH, AMT and DLD (dihydrolipoamide dehydrogenase), functions in mitochondrial folate metabolism ( a ) to cleave glycine, donating a one-carbon unit to THF. The Gldc GT1 allele ( b ) carries a gene-trap construct in intron 2. Splicing of exon2 to the gene-trap splice acceptor, SA, generates a truncated mRNA. The primers used for genotyping by <t>PCR</t> of genomic <t>DNA</t> are indicated ( b ). F1 and R3 generate a band in wild-type and heterozygous mice that is not detectable in homozygous Gldc GT1/GT1 mice ( c ). No PCR product is generated in +/+ samples when one or both primers are located within the trap construct (F2.R1 or F3.R2; full image of gel is included in Supplementary Information ). ( d ) Quantitative real time RT–PCR analysis of liver and brain samples at 5 weeks of age. ( e ) Enzymatic activity of the GCS in liver (* indicates significant difference from Gldc +/+ , P
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    Thermo Fisher easy dna genomic dna gdna
    Gene trap of Gldc ablates GCS activity. The GCS, comprising GLDC, GCSH, AMT and DLD (dihydrolipoamide dehydrogenase), functions in mitochondrial folate metabolism ( a ) to cleave glycine, donating a one-carbon unit to THF. The Gldc GT1 allele ( b ) carries a gene-trap construct in intron 2. Splicing of exon2 to the gene-trap splice acceptor, SA, generates a truncated mRNA. The primers used for genotyping by <t>PCR</t> of genomic <t>DNA</t> are indicated ( b ). F1 and R3 generate a band in wild-type and heterozygous mice that is not detectable in homozygous Gldc GT1/GT1 mice ( c ). No PCR product is generated in +/+ samples when one or both primers are located within the trap construct (F2.R1 or F3.R2; full image of gel is included in Supplementary Information ). ( d ) Quantitative real time RT–PCR analysis of liver and brain samples at 5 weeks of age. ( e ) Enzymatic activity of the GCS in liver (* indicates significant difference from Gldc +/+ , P
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    Thermo Fisher chargeswitch genomic dna gdna mini bacteria kit
    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it <t>DNA</t> ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.
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    92
    Thermo Fisher chargeswitch gdna
    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it <t>DNA</t> ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.
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    92
    Thermo Fisher transscript one step genomic dna gdna removal
    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it <t>DNA</t> ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.
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    90
    Thermo Fisher genomic dna fragmentation genomic dna gdna
    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it <t>DNA</t> ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.
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    93
    Thermo Fisher genecatcher gdna blood kits
    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it <t>DNA</t> ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.
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    Thermo Fisher magjet genomic dna kit
    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it <t>DNA</t> ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.
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    Image Search Results


    CRISPR/Cas9-mediated editing of the CFTR gene reduces the expression of CFTR. Ai : T7 endonuclease I (T7EI)-digested heteroduplexed PCR products, which contained indels (insertions/deletions) near the predicted lengths (228 and 168 bp), that were amplified from genomic DNA extracted from single amacrine cells transfected with pCRISPR-CFTR and pSpCas9. T7EI only digested heteroduplexed PCR products amplified from the amacrine cell transfected with pCRISPR-CFTR. Aii : T7EI-digested heteroduplexed PCR products, containing indels near the predicted lengths (228 and 168 bp), that were amplified from genomic DNA extracted from a population of cultured retinal cells transfected with pCRISPR-CFTRtd. T7EI did not digest heteroduplexed PCR products amplified from genomic DNA extracted from a population of cells transfected with pSpCas9td. B–G : representative amacrine cells transfected with pSpCas9td ( B–D ) and pCRISPR-CFTRtd ( E–G ). Anti-CFTR labeling ( C and F ) and merged images ( D and G ) are shown. White boxes in B–G are shown at higher magnification in Bi–Gi , respectively. Scale bars are 10 µm ( B–G ) and 5 µm ( Bi–Gi ). H–K : data from analyzed processes from cells transfected with the pSPCas9td construct and processes from cells transfected with pCRISPR-CFTRtd. Shown are mean total CFTR area ( H ), the mean CFTR object size ( I ), the mean CFTR intensity ( J ), and the mean tdTomato intensity within the CFTR object domains ( K ); n = 10 for both groups; A.U., arbitrary units. ** P

    Journal: Journal of Neurophysiology

    Article Title: A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl− from acidic organelles in amacrine cells

    doi: 10.1152/jn.00511.2017

    Figure Lengend Snippet: CRISPR/Cas9-mediated editing of the CFTR gene reduces the expression of CFTR. Ai : T7 endonuclease I (T7EI)-digested heteroduplexed PCR products, which contained indels (insertions/deletions) near the predicted lengths (228 and 168 bp), that were amplified from genomic DNA extracted from single amacrine cells transfected with pCRISPR-CFTR and pSpCas9. T7EI only digested heteroduplexed PCR products amplified from the amacrine cell transfected with pCRISPR-CFTR. Aii : T7EI-digested heteroduplexed PCR products, containing indels near the predicted lengths (228 and 168 bp), that were amplified from genomic DNA extracted from a population of cultured retinal cells transfected with pCRISPR-CFTRtd. T7EI did not digest heteroduplexed PCR products amplified from genomic DNA extracted from a population of cells transfected with pSpCas9td. B–G : representative amacrine cells transfected with pSpCas9td ( B–D ) and pCRISPR-CFTRtd ( E–G ). Anti-CFTR labeling ( C and F ) and merged images ( D and G ) are shown. White boxes in B–G are shown at higher magnification in Bi–Gi , respectively. Scale bars are 10 µm ( B–G ) and 5 µm ( Bi–Gi ). H–K : data from analyzed processes from cells transfected with the pSPCas9td construct and processes from cells transfected with pCRISPR-CFTRtd. Shown are mean total CFTR area ( H ), the mean CFTR object size ( I ), the mean CFTR intensity ( J ), and the mean tdTomato intensity within the CFTR object domains ( K ); n = 10 for both groups; A.U., arbitrary units. ** P

    Article Snippet: To detect indels (insertions/deletions) in the 7th exon of CFTR by nonhomologous end joining induced by Cas9-mediated double-stranded DNA cleavage, genomic DNA (gDNA) from single transfected dishes or single transfected amacrine cells was isolated using the Arcturus PicoPure kit (ThermoFisher Scientific).

    Techniques: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Transfection, Cell Culture, Labeling, Construct

    L1 hybridization enrichment strategy. A: Target site design. Schematic showing primary target site primers (TSPs, arrows) and secondary TSPs (bracketed arrows). The primary PCR amplifies a 5-kb empty target site. B: L1 amplification and hybridization enrichment. (1) A single filled site L1-containing molecule is present in a huge excess of empty site molecules. (2) Following primary PCR amplification, L1-containing amplicons are annealed to biotinylated L1-specific oligonucleotides (bio-oligos). (3) L1-containing amplicons are captured on streptavidin-coated paramagnetic beads. (4) L1-containing single-stranded DNA is released by thermal denaturation from the bead-bound bio-oligos. C: Screening enriched eluates for L1-containing targets. Full-length target molecules are amplified using primary TSPs (PCR1), then reamplified using appropriate combinations of an L1-specific primer together with a nested secondary TSP (bracketed) to target the L1/genomic DNA junction fragment, depending on the orientation of the insertion (PCR 2a or 2b). This nesting strategy prevents these amplicons becoming recoverable contaminants in subsequent MP-HE experiments.

    Journal: Human Mutation

    Article Title: L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline

    doi: 10.1002/humu.21533

    Figure Lengend Snippet: L1 hybridization enrichment strategy. A: Target site design. Schematic showing primary target site primers (TSPs, arrows) and secondary TSPs (bracketed arrows). The primary PCR amplifies a 5-kb empty target site. B: L1 amplification and hybridization enrichment. (1) A single filled site L1-containing molecule is present in a huge excess of empty site molecules. (2) Following primary PCR amplification, L1-containing amplicons are annealed to biotinylated L1-specific oligonucleotides (bio-oligos). (3) L1-containing amplicons are captured on streptavidin-coated paramagnetic beads. (4) L1-containing single-stranded DNA is released by thermal denaturation from the bead-bound bio-oligos. C: Screening enriched eluates for L1-containing targets. Full-length target molecules are amplified using primary TSPs (PCR1), then reamplified using appropriate combinations of an L1-specific primer together with a nested secondary TSP (bracketed) to target the L1/genomic DNA junction fragment, depending on the orientation of the insertion (PCR 2a or 2b). This nesting strategy prevents these amplicons becoming recoverable contaminants in subsequent MP-HE experiments.

    Article Snippet: Routine PCR A total of 20 µl PCR reactions contained 20 to 50 ng of genomic DNA (gDNA), PCR buffer (45 mM Tris-HCl pH 8.8, 11 mM ammonium sulphate, 5 mM MgCl2 , 6.7 mM 2-mercaptoethanol, 113 µg/ml BSA, 1.1 mM dNTPs), 0.05 µM of each primer and 0.025 U/µl 20:1 Taq /Pfu polymerases (Abgene, Epsom, UK; Stratagene, La Jolla, CA).

    Techniques: Hybridization, Polymerase Chain Reaction, Amplification

    Amplification of a full-length L1 insertion in a multiplex PCR. Genomic DNA from donor A, heterozygous for the polymorphic AL121819 L1 insertion, was amplified using primers for all 10 target loci (10-plex, rightmost lanes) or for the 10 target loci plus the AL121819 insertion (11-plex, leftmost lanes). PCR products were analysed by agarose gel electrophoresis. DNA−, negative control; M, 1-kb DNA ladder (NEB). The 11-plex PCR shows two additional products (arrowed) corresponding to the empty AL121819 allele (6 kb) and the filled allele (12 kb).

    Journal: Human Mutation

    Article Title: L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline

    doi: 10.1002/humu.21533

    Figure Lengend Snippet: Amplification of a full-length L1 insertion in a multiplex PCR. Genomic DNA from donor A, heterozygous for the polymorphic AL121819 L1 insertion, was amplified using primers for all 10 target loci (10-plex, rightmost lanes) or for the 10 target loci plus the AL121819 insertion (11-plex, leftmost lanes). PCR products were analysed by agarose gel electrophoresis. DNA−, negative control; M, 1-kb DNA ladder (NEB). The 11-plex PCR shows two additional products (arrowed) corresponding to the empty AL121819 allele (6 kb) and the filled allele (12 kb).

    Article Snippet: Routine PCR A total of 20 µl PCR reactions contained 20 to 50 ng of genomic DNA (gDNA), PCR buffer (45 mM Tris-HCl pH 8.8, 11 mM ammonium sulphate, 5 mM MgCl2 , 6.7 mM 2-mercaptoethanol, 113 µg/ml BSA, 1.1 mM dNTPs), 0.05 µM of each primer and 0.025 U/µl 20:1 Taq /Pfu polymerases (Abgene, Epsom, UK; Stratagene, La Jolla, CA).

    Techniques: Amplification, Multiplex Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control

    Multiplex PCR amplification of target loci. All 10 target loci were amplified from genomic DNA in a 10-plex PCR reaction. PCR products were analyzed by agarose gel electrophoresis, before or after digestion with Bss SI, as indicated. Amplicon sizes are shown in Table 1 . DNA−, negative control reaction with no genomic DNA. Target identities in the Bss SI digest are shown using the identifiers in Table 1 . Targets FKTN and HBB are not fully resolved but show approximately doubled band intensity, as expected for two comigrating fragments. This is also the case for the RP2 and DMD targets.

    Journal: Human Mutation

    Article Title: L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline

    doi: 10.1002/humu.21533

    Figure Lengend Snippet: Multiplex PCR amplification of target loci. All 10 target loci were amplified from genomic DNA in a 10-plex PCR reaction. PCR products were analyzed by agarose gel electrophoresis, before or after digestion with Bss SI, as indicated. Amplicon sizes are shown in Table 1 . DNA−, negative control reaction with no genomic DNA. Target identities in the Bss SI digest are shown using the identifiers in Table 1 . Targets FKTN and HBB are not fully resolved but show approximately doubled band intensity, as expected for two comigrating fragments. This is also the case for the RP2 and DMD targets.

    Article Snippet: Routine PCR A total of 20 µl PCR reactions contained 20 to 50 ng of genomic DNA (gDNA), PCR buffer (45 mM Tris-HCl pH 8.8, 11 mM ammonium sulphate, 5 mM MgCl2 , 6.7 mM 2-mercaptoethanol, 113 µg/ml BSA, 1.1 mM dNTPs), 0.05 µM of each primer and 0.025 U/µl 20:1 Taq /Pfu polymerases (Abgene, Epsom, UK; Stratagene, La Jolla, CA).

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Negative Control

    Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C.

    Journal: Human Mutation

    Article Title: L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline

    doi: 10.1002/humu.21533

    Figure Lengend Snippet: Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C.

    Article Snippet: Routine PCR A total of 20 µl PCR reactions contained 20 to 50 ng of genomic DNA (gDNA), PCR buffer (45 mM Tris-HCl pH 8.8, 11 mM ammonium sulphate, 5 mM MgCl2 , 6.7 mM 2-mercaptoethanol, 113 µg/ml BSA, 1.1 mM dNTPs), 0.05 µM of each primer and 0.025 U/µl 20:1 Taq /Pfu polymerases (Abgene, Epsom, UK; Stratagene, La Jolla, CA).

    Techniques: Hybridization, Multiplex Assay, Polymerase Chain Reaction, Labeling, Derivative Assay, Negative Control, Produced

    Comparison of DNA extraction methods. (A) Experimental design. Human feces, pig feces, and hospital sewage were extracted using seven different DNA extraction methods ( Table 1 ): InnuPure C16, MagNA Pure LC DNA isolation kit III, Easy-DNA gDNA purification kit, MP FastDNA Spin kit, PowerSoil DNA isolation kit, QIAamp DNA stool minikit, and QIAamp DNA stool minikit plus bead beating (for details, see Materials and Methods). DNA concentration, purity, and stability were examined, and microbial community composition was determined using 16S rRNA gene profiling and metagenomics (selected samples). (B) DNA from each method was dissolved in 100 µl solution, and DNA concentrations were determined using Qubit dsDNA BR assay kit measurements. Values represent averages from duplicate or triplicate DNA extractions (see also Table S1A in the supplemental material). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (see also Table S1B ).

    Journal: mSystems

    Article Title: Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

    doi: 10.1128/mSystems.00095-16

    Figure Lengend Snippet: Comparison of DNA extraction methods. (A) Experimental design. Human feces, pig feces, and hospital sewage were extracted using seven different DNA extraction methods ( Table 1 ): InnuPure C16, MagNA Pure LC DNA isolation kit III, Easy-DNA gDNA purification kit, MP FastDNA Spin kit, PowerSoil DNA isolation kit, QIAamp DNA stool minikit, and QIAamp DNA stool minikit plus bead beating (for details, see Materials and Methods). DNA concentration, purity, and stability were examined, and microbial community composition was determined using 16S rRNA gene profiling and metagenomics (selected samples). (B) DNA from each method was dissolved in 100 µl solution, and DNA concentrations were determined using Qubit dsDNA BR assay kit measurements. Values represent averages from duplicate or triplicate DNA extractions (see also Table S1A in the supplemental material). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (see also Table S1B ).

    Article Snippet: In a first step, seven DNA isolation procedures were examined, namely, InnuPure C16 from Analytic Jena AG (InnuPure), MagNA Pure LC DNA isolation kit III from Roche (MagNA Pure), Easy-DNA genomic DNA (gDNA) purification kit from Invitrogen (Easy-DNA), MP FastDNA Spin kit from MP Biomedicals (FastDNA), PowerSoil DNA isolation kit from MoBio (PowerSoil.HMP), QIAamp DNA stool minikit from Qiagen (QIAStool), and QIAamp DNA stool minikit plus bead beating from Qiagen (QIAStool+BB) ( and details below).

    Techniques: DNA Extraction, Purification, Concentration Assay, Sequencing

    Gene trap of Gldc ablates GCS activity. The GCS, comprising GLDC, GCSH, AMT and DLD (dihydrolipoamide dehydrogenase), functions in mitochondrial folate metabolism ( a ) to cleave glycine, donating a one-carbon unit to THF. The Gldc GT1 allele ( b ) carries a gene-trap construct in intron 2. Splicing of exon2 to the gene-trap splice acceptor, SA, generates a truncated mRNA. The primers used for genotyping by PCR of genomic DNA are indicated ( b ). F1 and R3 generate a band in wild-type and heterozygous mice that is not detectable in homozygous Gldc GT1/GT1 mice ( c ). No PCR product is generated in +/+ samples when one or both primers are located within the trap construct (F2.R1 or F3.R2; full image of gel is included in Supplementary Information ). ( d ) Quantitative real time RT–PCR analysis of liver and brain samples at 5 weeks of age. ( e ) Enzymatic activity of the GCS in liver (* indicates significant difference from Gldc +/+ , P

    Journal: Nature Communications

    Article Title: Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice

    doi: 10.1038/ncomms7388

    Figure Lengend Snippet: Gene trap of Gldc ablates GCS activity. The GCS, comprising GLDC, GCSH, AMT and DLD (dihydrolipoamide dehydrogenase), functions in mitochondrial folate metabolism ( a ) to cleave glycine, donating a one-carbon unit to THF. The Gldc GT1 allele ( b ) carries a gene-trap construct in intron 2. Splicing of exon2 to the gene-trap splice acceptor, SA, generates a truncated mRNA. The primers used for genotyping by PCR of genomic DNA are indicated ( b ). F1 and R3 generate a band in wild-type and heterozygous mice that is not detectable in homozygous Gldc GT1/GT1 mice ( c ). No PCR product is generated in +/+ samples when one or both primers are located within the trap construct (F2.R1 or F3.R2; full image of gel is included in Supplementary Information ). ( d ) Quantitative real time RT–PCR analysis of liver and brain samples at 5 weeks of age. ( e ) Enzymatic activity of the GCS in liver (* indicates significant difference from Gldc +/+ , P

    Article Snippet: qRT–PCR RNA was prepared using TRI reagent (Invitrogen), genomic DNA removed by DNase I digestion (DNA-free, Ambion) and first strand cDNA synthesis performed using random hexamers (Superscript VILO cDNA synthesis kit).

    Techniques: Activity Assay, Construct, Polymerase Chain Reaction, Mouse Assay, Generated, Quantitative RT-PCR

    Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it DNA ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.

    Journal: mSphere

    Article Title: Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

    doi: 10.1128/mSphere.00100-15

    Figure Lengend Snippet: Genetic disruption of the bont /B2 gene in strain CDC69016. (A) Schematic presentation of the wild-type (top) and mutated (bottom) botulinum neurotoxin B2 gene. The group II intron is shown as a black wide arrow inserted on the sense strand of the toxin gene (gray arrow) between nucleotides 381 and 382 as indicated by the vertical black arrows. The white arrow inside the intron element in the opposite orientation to the intron and toxin gene is a retrotransposition-activated erythromycin (RAM-Erm) resistance gene. The locations of the forward (F) and reverse (R) PCR primers are shown with horizontal arrows on either side of the intron insertion site. The expected size of the PCR products for the wild-type (WT) strain is 238 bp, and it is 2,086 bp for the inactivated BoNT/B2 gene. (B) PCR products of five putative mutant clones (clones 1 to 5) and two samples of the wild-type (WT) strain. The positions of molecular size markers (M) (Track-it DNA ladder [Life Technologies]) in base pairs are shown to the right of the gel. (C and D) Southern hybridization with the intron probe (erythromycin gene) (C) and ethidium bromide-stained 1% agarose gel of genomic DNA digested with restriction enzyme HindIII (D). Lanes 1 to 5, five individual putative BoNT/B2 mutant clones; lane 6, wild-type CDC69016 strain; lane 7, C. botulinum strain CDC-A3 as a negative control; lanes 8 and 9, DNA markers (NEB, Ipswich, MA). The sizes of the DNA markers (in base pairs) are indicated to the right of the gel.

    Article Snippet: Genomic DNA was isolated from the wild-type C. botulinum strain CDC69016 and the CDC69016/B2tox− mutant using the ChargeSwitch genomic DNA (gDNA) Mini Bacteria kit (Life Technologies, Carlsbad, CA) following the manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction, Mutagenesis, Clone Assay, Hybridization, Staining, Agarose Gel Electrophoresis, Negative Control