restriction enzymes ecori  (New England Biolabs)


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    EcoRI
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    EcoRI 50 000 units
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    r0101l
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    Restriction Enzymes
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    New England Biolabs restriction enzymes ecori
    EcoRI
    EcoRI 50 000 units
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    Images

    1) Product Images from "Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences"

    Article Title: Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

    Journal: Journal of industrial microbiology & biotechnology

    doi: 10.1007/s10295-018-2049-x

    The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown
    Figure Legend Snippet: The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Techniques Used: Plasmid Preparation, Derivative Assay, Purification, Isolation, Transformation Assay, Polymerase Chain Reaction, Molecular Weight

    2) Product Images from "Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol"

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106713

    Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.
    Figure Legend Snippet: Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Techniques Used: Sequencing

    Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).
    Figure Legend Snippet: Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Techniques Used: Sequencing, Isolation, Agarose Gel Electrophoresis

    3) Product Images from "Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine"

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    Journal: Mutagenesis

    doi: 10.1093/mutage/get027

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).
    Figure Legend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Techniques Used: Southern Blot, Incubation, Generated

    4) Product Images from "Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates"

    Article Title: Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates

    Journal: Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology

    doi: 10.1007/s12639-014-0582-8

    RE digestion of TE-PCR product with EcoRI, Pst I, Eco91l and HindIII . [ Lane M 100 bp DNA ladder; Lane 1 TE-PCR product with out RE; Lane 2 TE-PCR product with EcoRI ; Lane 3 TE-PCR product with Pst I ; Lane 5 TE-PCR product with out RE; Lane 6
    Figure Legend Snippet: RE digestion of TE-PCR product with EcoRI, Pst I, Eco91l and HindIII . [ Lane M 100 bp DNA ladder; Lane 1 TE-PCR product with out RE; Lane 2 TE-PCR product with EcoRI ; Lane 3 TE-PCR product with Pst I ; Lane 5 TE-PCR product with out RE; Lane 6

    Techniques Used: Polymerase Chain Reaction

    5) Product Images from "Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino"

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17101690

    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
    Figure Legend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Techniques Used: DNA Methylation Assay, Methylation

    6) Product Images from "Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker"

    Article Title: Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker

    Journal: BMC Veterinary Research

    doi: 10.1186/1746-6148-7-80

    Infection/transfection scheme for generation of recombinant ORFV . A . Construction of the recombinant transfer vector pSPV-EGFP. A cassette of selectable markers of E.coli neo and gusA genes in pZIPPY-neo/gus vector was replace by the EGFP reporter gene amplified from pEGFP-N1 vector (Clontech, CA) to generate the recombinant vector pSPV-EGFP. B . Generation of recombinant ORFV. OFTU Cells are infected with OV-IA82 and transfected with the transfer vector pSPV-113LF-EGFP-113RF. The resultant virus mixture is then plated on OFTu cells to eliminate OV-IA82 and the desired viruses were isolated. MCS: Multiple cloning sites. B: BglII; E: EcoRI; H: HindIII; N: NotI; and X: XhoI. V: vaccinia virus (strain WR) VV early/late protomer VVp7.5. L: Up stream of ORFV113 un-transcription region; R: Down stream of ORFV113 un-transcription region. DHR: double homologous recombination.
    Figure Legend Snippet: Infection/transfection scheme for generation of recombinant ORFV . A . Construction of the recombinant transfer vector pSPV-EGFP. A cassette of selectable markers of E.coli neo and gusA genes in pZIPPY-neo/gus vector was replace by the EGFP reporter gene amplified from pEGFP-N1 vector (Clontech, CA) to generate the recombinant vector pSPV-EGFP. B . Generation of recombinant ORFV. OFTU Cells are infected with OV-IA82 and transfected with the transfer vector pSPV-113LF-EGFP-113RF. The resultant virus mixture is then plated on OFTu cells to eliminate OV-IA82 and the desired viruses were isolated. MCS: Multiple cloning sites. B: BglII; E: EcoRI; H: HindIII; N: NotI; and X: XhoI. V: vaccinia virus (strain WR) VV early/late protomer VVp7.5. L: Up stream of ORFV113 un-transcription region; R: Down stream of ORFV113 un-transcription region. DHR: double homologous recombination.

    Techniques Used: Infection, Transfection, Recombinant, Plasmid Preparation, Amplification, Isolation, Clone Assay, Homologous Recombination

    7) Product Images from "Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon"

    Article Title: Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon

    Journal: Virology Journal

    doi: 10.1186/1743-422X-6-173

    Construction and evaluation of a SAV3 based replicon . A) A SAV3 replicon is launched from the CMV promoter (red arrow) in the pVAX1 backbone, and transcription stops at the BGH polyA signal (red box). A synthetic DNA encoding a hammerhead ribozyme was fused to the SAV3 5'-UTR and a polyadenylated tail was fused to the 3'-UTR. The ORF encoding the SAV3 structural proteins was replaced by an ORF encoding EGFP inserted between introduced AgeI and AscI sites. Position of the EcoRI site used for restriction enzyme analysis is indicated. B) Restriction enzyme analysis by digestion of pmSAV3 with EcoRI, AgeI and AscI. Lane 1: Smartladder (Eurogentec). Lane 2: pmSAV3 after triple digest with EcoRI, AgeI and AscI. Bands corresponding to the pVAX1 backbone, nsP coding sequence and EGFP coding sequence are indicated. C) Expression of EGFP in BF2 cells after transfection with pmSAV3. Both CHSE and BF2 cells facilitated successful expression of the EGFP reporter. EGFP expression became visible from day 2 p.t. in CHSE cells and 3 d.p.t. in BF2 cells.
    Figure Legend Snippet: Construction and evaluation of a SAV3 based replicon . A) A SAV3 replicon is launched from the CMV promoter (red arrow) in the pVAX1 backbone, and transcription stops at the BGH polyA signal (red box). A synthetic DNA encoding a hammerhead ribozyme was fused to the SAV3 5'-UTR and a polyadenylated tail was fused to the 3'-UTR. The ORF encoding the SAV3 structural proteins was replaced by an ORF encoding EGFP inserted between introduced AgeI and AscI sites. Position of the EcoRI site used for restriction enzyme analysis is indicated. B) Restriction enzyme analysis by digestion of pmSAV3 with EcoRI, AgeI and AscI. Lane 1: Smartladder (Eurogentec). Lane 2: pmSAV3 after triple digest with EcoRI, AgeI and AscI. Bands corresponding to the pVAX1 backbone, nsP coding sequence and EGFP coding sequence are indicated. C) Expression of EGFP in BF2 cells after transfection with pmSAV3. Both CHSE and BF2 cells facilitated successful expression of the EGFP reporter. EGFP expression became visible from day 2 p.t. in CHSE cells and 3 d.p.t. in BF2 cells.

    Techniques Used: Sequencing, Expressing, Transfection

    8) Product Images from "Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli"

    Article Title: Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-4-36

    Sequence of the polylinker . The polylinker for pB42-C1 is shown. Restriction sites are underlined and labeled. The sequence of pB42-C2 and pB42-C3 are identical except for the addition of one and two additional G residues immediately prior to the EcoRI site, as indicated.
    Figure Legend Snippet: Sequence of the polylinker . The polylinker for pB42-C1 is shown. Restriction sites are underlined and labeled. The sequence of pB42-C2 and pB42-C3 are identical except for the addition of one and two additional G residues immediately prior to the EcoRI site, as indicated.

    Techniques Used: Sequencing, Labeling

    9) Product Images from "The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex"

    Article Title: The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex

    Journal: eLife

    doi: 10.7554/eLife.11721

    Reconstituted nucleosomes on restricted λdsDNA: EcoRI digestion generated l-DNA fragments of 21 kbp, 7.5 kbp, 5.8 kbp, 5.6 kbp, 4.8 kbp, and 3.5 kbp. DOI: http://dx.doi.org/10.7554/eLife.11721.015
    Figure Legend Snippet: Reconstituted nucleosomes on restricted λdsDNA: EcoRI digestion generated l-DNA fragments of 21 kbp, 7.5 kbp, 5.8 kbp, 5.6 kbp, 4.8 kbp, and 3.5 kbp. DOI: http://dx.doi.org/10.7554/eLife.11721.015

    Techniques Used: Generated

    10) Product Images from "Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere"

    Article Title: Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere

    Journal:

    doi: 10.1128/AEM.71.12.8537-8547.2005

    Schematic diagram of the construction of the pNice fusion plasmid containing the L28H- fnr gene. Cm r , Km r , and Ap r , resistance to chloramphenicol, kanamycin, and ampicillin, respectively. narGp indicates a 592-bp HindIII-EcoRI fragment of the narG promoter-regulatory
    Figure Legend Snippet: Schematic diagram of the construction of the pNice fusion plasmid containing the L28H- fnr gene. Cm r , Km r , and Ap r , resistance to chloramphenicol, kanamycin, and ampicillin, respectively. narGp indicates a 592-bp HindIII-EcoRI fragment of the narG promoter-regulatory

    Techniques Used: Plasmid Preparation

    11) Product Images from "Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters"

    Article Title: Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt731

    Duplexes carrying damaged sites used in this study. Oligonucleotides carrying oG, hU and U. The modified bases are indicated in bold. oG was separated by 3 bp from hU located on the complementary strand in MDS/+1, MDS/−5. Uracil was positioned at +1 position relatively to hU in MDS/+1 and at −5 position in MDS/−5. The control U/U oligonucleotide consists of two bistranded Us separated by 5 bp. Undamaged and MDS-containing 56-mers duplexes harbor EcoRI and XhoI restriction sites at each extremity as indicated.
    Figure Legend Snippet: Duplexes carrying damaged sites used in this study. Oligonucleotides carrying oG, hU and U. The modified bases are indicated in bold. oG was separated by 3 bp from hU located on the complementary strand in MDS/+1, MDS/−5. Uracil was positioned at +1 position relatively to hU in MDS/+1 and at −5 position in MDS/−5. The control U/U oligonucleotide consists of two bistranded Us separated by 5 bp. Undamaged and MDS-containing 56-mers duplexes harbor EcoRI and XhoI restriction sites at each extremity as indicated.

    Techniques Used: Modification

    12) Product Images from "Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases"

    Article Title: Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0494-2

    Workflow of TALEN-mediated generation of a monkey embryo carrying an EmGFP reporter in the OCT4 gene. TALENs-coding plasmids, pTALEN-Maca-oct4-E1-F/R, and the donor vector Donor-E1-PKID-EmGFP that targets exon 1 of the OCT4 gene were designed and co-injected into the cytoplasm of a zygote 6–8 h after fertilization. Treated embryos at the blastocyst, morula and 16-cell stages were collected and analyzed
    Figure Legend Snippet: Workflow of TALEN-mediated generation of a monkey embryo carrying an EmGFP reporter in the OCT4 gene. TALENs-coding plasmids, pTALEN-Maca-oct4-E1-F/R, and the donor vector Donor-E1-PKID-EmGFP that targets exon 1 of the OCT4 gene were designed and co-injected into the cytoplasm of a zygote 6–8 h after fertilization. Treated embryos at the blastocyst, morula and 16-cell stages were collected and analyzed

    Techniques Used: TALENs, Plasmid Preparation, Injection

    13) Product Images from "Splicing-Mediated Autoregulation Modulates Rpl22p Expression in Saccharomyces cerevisiae"

    Article Title: Splicing-Mediated Autoregulation Modulates Rpl22p Expression in Saccharomyces cerevisiae

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005999

    Identification of a regulatory element necessary for binding of Rpl22 to the unspliced RPL22B mRNP in vivo . A. Left : Schematic of RPL22B intron reporter system based on a pUG35 plasmid backbone. Also indicated are the deletions of the binary search. Deletions highlighted in bold purple font indicate those that result in a loss of splicing inhibition. Right : Northern blots demonstrating the loss of splicing inhibition upon deletion of certain segments of the RPL22B intron. SCR1 was used as a loading control. Note that all constructs appearing in these blots do not contain the alternative 5’-splice site. See S1 Text for details. B. Structure of the RPL22B intron bounded by nucleotides G-153 and C-246 as predicted by Mfold. Regions of interest have been denoted. Nucleotides of the lower distal stem and nucleotides U221 through A224 of the putative RNA Internal Loop were experimentally determined to be important for autoregulatory activity and are colored orange and green, respectively. C. Experimentally deduced intronic regulatory element that mediates the inhibition of splicing of the RPL22B pre-mRNA. Nucleotide colors from Panel B are retained for comparison. Details describing the experiments leading to the inferred secondary structure are provided in S1 Text . D. RNA immunoprecipitation shows the association of Rpl22p with the unspliced RPL22B mRNP that requires the regulatory element. Upper panel : RT-PCR analysis of RPL22B transcripts derived from cells expressing untagged Rpl22Ap (-), Rpl22Ap with the 13-Myc epitope tag (+), and cells with the RPL22B intronic regulatory element deleted (Δ) and expressing tagged protein (+). Total input RNA and immunoprecipitated RNA were analyzed from each strain. Labeled bands show the unspliced (US) and spliced (S) species. Lower panel : RT-PCR analysis of TFC3 transcripts performed by similar methods as described for panel A. Bands indicate the unspliced (US) and spliced (S) species. The gel images have been truncated for space considerations. Full, unattenuated versions of the gels can be viewed in S8B Fig .
    Figure Legend Snippet: Identification of a regulatory element necessary for binding of Rpl22 to the unspliced RPL22B mRNP in vivo . A. Left : Schematic of RPL22B intron reporter system based on a pUG35 plasmid backbone. Also indicated are the deletions of the binary search. Deletions highlighted in bold purple font indicate those that result in a loss of splicing inhibition. Right : Northern blots demonstrating the loss of splicing inhibition upon deletion of certain segments of the RPL22B intron. SCR1 was used as a loading control. Note that all constructs appearing in these blots do not contain the alternative 5’-splice site. See S1 Text for details. B. Structure of the RPL22B intron bounded by nucleotides G-153 and C-246 as predicted by Mfold. Regions of interest have been denoted. Nucleotides of the lower distal stem and nucleotides U221 through A224 of the putative RNA Internal Loop were experimentally determined to be important for autoregulatory activity and are colored orange and green, respectively. C. Experimentally deduced intronic regulatory element that mediates the inhibition of splicing of the RPL22B pre-mRNA. Nucleotide colors from Panel B are retained for comparison. Details describing the experiments leading to the inferred secondary structure are provided in S1 Text . D. RNA immunoprecipitation shows the association of Rpl22p with the unspliced RPL22B mRNP that requires the regulatory element. Upper panel : RT-PCR analysis of RPL22B transcripts derived from cells expressing untagged Rpl22Ap (-), Rpl22Ap with the 13-Myc epitope tag (+), and cells with the RPL22B intronic regulatory element deleted (Δ) and expressing tagged protein (+). Total input RNA and immunoprecipitated RNA were analyzed from each strain. Labeled bands show the unspliced (US) and spliced (S) species. Lower panel : RT-PCR analysis of TFC3 transcripts performed by similar methods as described for panel A. Bands indicate the unspliced (US) and spliced (S) species. The gel images have been truncated for space considerations. Full, unattenuated versions of the gels can be viewed in S8B Fig .

    Techniques Used: Binding Assay, In Vivo, Plasmid Preparation, Inhibition, Northern Blot, Construct, Activity Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Labeling

    14) Product Images from "Prokaryotic expression of MLAA-34 and generation of a novel human ScFv against MLAA-34 by phage display technology"

    Article Title: Prokaryotic expression of MLAA-34 and generation of a novel human ScFv against MLAA-34 by phage display technology

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16590

    Construction of the MLAA-34 expression vector (A) PET 28a Vector information. Component sequence: T7 promoter-6His-MCS Clone sites: EcoRI / SalI. (B) The amplified MLAA-34 fragment was cloned into the PET-28a vector, and positive transformation was identified by PCR; the expected product size was 728 bp. Lane 1: negative control (ddH2O); lane 2: negative control (self-connected control group); lane 3: positive control (GAPDH); lane 4: marker 5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500 bp, 250 bp, 100 bp; lanes 5-12: gene 1-8 transformation.
    Figure Legend Snippet: Construction of the MLAA-34 expression vector (A) PET 28a Vector information. Component sequence: T7 promoter-6His-MCS Clone sites: EcoRI / SalI. (B) The amplified MLAA-34 fragment was cloned into the PET-28a vector, and positive transformation was identified by PCR; the expected product size was 728 bp. Lane 1: negative control (ddH2O); lane 2: negative control (self-connected control group); lane 3: positive control (GAPDH); lane 4: marker 5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500 bp, 250 bp, 100 bp; lanes 5-12: gene 1-8 transformation.

    Techniques Used: Expressing, Plasmid Preparation, Positron Emission Tomography, Sequencing, Amplification, Clone Assay, Transformation Assay, Polymerase Chain Reaction, Negative Control, Positive Control, Marker

    15) Product Images from "High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis"

    Article Title: High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis

    Journal: Genome Research

    doi:

    Southern blot analysis of BamHI- and EcoRI-digested BAC DNA identifies six copies of Naip exon 11 and five copies of Naip exon 3. Correlation of the restriction fragments with specific Naip loci was done by comparison of the bands on the BAC blot with predicted fragments from genomic sequence and our previous physical map of the 129 Naip ). Horizontal bars and numbers indicate position and size (kb) of fragments in 1-kb ladder molecular weight marker (GIBCO). ( A ) BamHI-digested DNA probed with Naip exon 11 identifies six Naip exon 11 loci. 129 haplotype genomic sequence predicts the observed 14.3-kb fragment mapping to Naip1 , a 9-kb fragment mapping to Naip2 , an 8.6-kb fragment mapping to Naip5 , and a doublet of 3.5 kb and 3.6 kb mapping to Naip6 and Naip3 . The remaining 2.2-kb band maps to Δ Naip , as observed in the 129 haplotype (data not shown). Asterisk indicates vector junction fragments mapping to Naip1 and Naip2. ( B ) EcoRI-digested DN A probed with Naip exon 3 identifies five Naip exon 3 loci. 129 haplotype genomic sequence predicts the observed 10.2-kb fragment mapping to Naip5 , an 8.5-kb fragment mapping to Naip2 , a 7.5-kb fragment mapping to Naip1 , a 7.2-kb fragment mapping to Naip6 , and 2.1-kb mapping to Naip3 .
    Figure Legend Snippet: Southern blot analysis of BamHI- and EcoRI-digested BAC DNA identifies six copies of Naip exon 11 and five copies of Naip exon 3. Correlation of the restriction fragments with specific Naip loci was done by comparison of the bands on the BAC blot with predicted fragments from genomic sequence and our previous physical map of the 129 Naip ). Horizontal bars and numbers indicate position and size (kb) of fragments in 1-kb ladder molecular weight marker (GIBCO). ( A ) BamHI-digested DNA probed with Naip exon 11 identifies six Naip exon 11 loci. 129 haplotype genomic sequence predicts the observed 14.3-kb fragment mapping to Naip1 , a 9-kb fragment mapping to Naip2 , an 8.6-kb fragment mapping to Naip5 , and a doublet of 3.5 kb and 3.6 kb mapping to Naip6 and Naip3 . The remaining 2.2-kb band maps to Δ Naip , as observed in the 129 haplotype (data not shown). Asterisk indicates vector junction fragments mapping to Naip1 and Naip2. ( B ) EcoRI-digested DN A probed with Naip exon 3 identifies five Naip exon 3 loci. 129 haplotype genomic sequence predicts the observed 10.2-kb fragment mapping to Naip5 , an 8.5-kb fragment mapping to Naip2 , a 7.5-kb fragment mapping to Naip1 , a 7.2-kb fragment mapping to Naip6 , and 2.1-kb mapping to Naip3 .

    Techniques Used: Southern Blot, BAC Assay, Sequencing, Molecular Weight, Marker, Plasmid Preparation

    16) Product Images from "Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate "

    Article Title: Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01621-07

    Southern analysis of strains M129 and S1. Chromosomal DNA isolated from M129 (lanes 1, 3, 5, and 7) and S1 (lanes 2, 4, 6, and 8) was digested to completion with EcoRI and HindIII, and the fragments generated were separated on 1% agarose gels
    Figure Legend Snippet: Southern analysis of strains M129 and S1. Chromosomal DNA isolated from M129 (lanes 1, 3, 5, and 7) and S1 (lanes 2, 4, 6, and 8) was digested to completion with EcoRI and HindIII, and the fragments generated were separated on 1% agarose gels

    Techniques Used: Isolation, Generated

    17) Product Images from "Identification of Hedysarum Varieties Using Amplified Fragment Length Polymorphism on a Capillary Electrophoresis System"

    Article Title: Identification of Hedysarum Varieties Using Amplified Fragment Length Polymorphism on a Capillary Electrophoresis System

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    Final genotypes in a binary format for subset of the samples and alleles from the AFLP run with the primer pair FAM-Ecori-ACA and Msei-CTT as displayed in the Genotypes tab of the GeneMapper software.
    Figure Legend Snippet: Final genotypes in a binary format for subset of the samples and alleles from the AFLP run with the primer pair FAM-Ecori-ACA and Msei-CTT as displayed in the Genotypes tab of the GeneMapper software.

    Techniques Used: Software

    18) Product Images from "Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications"

    Article Title: Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.062082606

    Fragment library distribution. ( A ) Fragment size distribution is unbiased. SYBR-Safe stained 1% agarose gel of 144 individual clones, generated by shotgun capture of the fragmentation reaction in the ligase-free cloning vector pCR-Blunt-II TOPO (Invitrogen). Clones were pooled in lots of 12 and miniprepped, and captured DNA inserts were released as EcoRI fragments, with 12 vector-derived bases still attached to each end. The distribution of fragment sizes populates the desired range 0.1–1.0 kb. ( B ) The fragment position is random. Coverage plot of 63 randomly selected and sequenced clones (black lines) from the p85α fragment library, ordered according to their 5′-end ( bottom to top ), arrayed against the 2175-bp sequence of human p85α. Apart from clones beginning at the actual 5′-end of the target gene, the start positions of the fragments are evenly distributed across the target gene, which is fully sampled. Although the sample size is far too small for statistical significance, it is fully consistent with random and unbiased fragmentation. ( C ) As B , but with the data sorted by 3′-end position. ( D ) Histogram of fragment size frequency (N). Fragment sizes are binned in intervals of 200 bp. Although the sample size is too small for statistical significance, the distribution is consistent with the expected Poisson distribution for a random fragmentation process.
    Figure Legend Snippet: Fragment library distribution. ( A ) Fragment size distribution is unbiased. SYBR-Safe stained 1% agarose gel of 144 individual clones, generated by shotgun capture of the fragmentation reaction in the ligase-free cloning vector pCR-Blunt-II TOPO (Invitrogen). Clones were pooled in lots of 12 and miniprepped, and captured DNA inserts were released as EcoRI fragments, with 12 vector-derived bases still attached to each end. The distribution of fragment sizes populates the desired range 0.1–1.0 kb. ( B ) The fragment position is random. Coverage plot of 63 randomly selected and sequenced clones (black lines) from the p85α fragment library, ordered according to their 5′-end ( bottom to top ), arrayed against the 2175-bp sequence of human p85α. Apart from clones beginning at the actual 5′-end of the target gene, the start positions of the fragments are evenly distributed across the target gene, which is fully sampled. Although the sample size is far too small for statistical significance, it is fully consistent with random and unbiased fragmentation. ( C ) As B , but with the data sorted by 3′-end position. ( D ) Histogram of fragment size frequency (N). Fragment sizes are binned in intervals of 200 bp. Although the sample size is too small for statistical significance, the distribution is consistent with the expected Poisson distribution for a random fragmentation process.

    Techniques Used: Staining, Agarose Gel Electrophoresis, Clone Assay, Generated, Plasmid Preparation, Polymerase Chain Reaction, Derivative Assay, Sequencing

    19) Product Images from "Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity"

    Article Title: Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-015-0041-x

    PCR-based lacZα -complementation system used to determine the fidelity of HIV RT. (A) An overview of the procedure used to assess polymerase fidelity is presented. RNA is represented by broken lines and DNA is represented by solid line. Primers have arrowheads at the 3′ end. The ~760 nt template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3′ and 5′ ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. The filled box at the bottom of the figure is the 115 base region of the lacZ α gene that was scored in the assay. Details for specific steps are provided under Materials and Methods. (B) Plasmid pBSM13ΔPvuII 1146 , is shown. Relevant sites on the plasmid are indicated and numbering is based on the parent plasmid (pBSM13+ (Stratagene)). (C) The nt and amino acid sequence for the 115 base region of the lacZ α gene that was scored in the assay is shown. Both strands of the DNA plasmid are shown since HIV RT synthesis was performed in both directions (see Figure 2A). A line is drawn above the 92 nts that are in the detectable area for substitution mutations while frameshifts can be detected over the entire 115 nt region. Based on a previous cataloging of mutations in this gene [ 51 ], the assay can detect 116 different substitutions (33.6% of the 345 possible substitutions in the 115 nt sequence) and 100% of the frameshift mutations.
    Figure Legend Snippet: PCR-based lacZα -complementation system used to determine the fidelity of HIV RT. (A) An overview of the procedure used to assess polymerase fidelity is presented. RNA is represented by broken lines and DNA is represented by solid line. Primers have arrowheads at the 3′ end. The ~760 nt template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3′ and 5′ ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. The filled box at the bottom of the figure is the 115 base region of the lacZ α gene that was scored in the assay. Details for specific steps are provided under Materials and Methods. (B) Plasmid pBSM13ΔPvuII 1146 , is shown. Relevant sites on the plasmid are indicated and numbering is based on the parent plasmid (pBSM13+ (Stratagene)). (C) The nt and amino acid sequence for the 115 base region of the lacZ α gene that was scored in the assay is shown. Both strands of the DNA plasmid are shown since HIV RT synthesis was performed in both directions (see Figure 2A). A line is drawn above the 92 nts that are in the detectable area for substitution mutations while frameshifts can be detected over the entire 115 nt region. Based on a previous cataloging of mutations in this gene [ 51 ], the assay can detect 116 different substitutions (33.6% of the 345 possible substitutions in the 115 nt sequence) and 100% of the frameshift mutations.

    Techniques Used: Polymerase Chain Reaction, DNA Synthesis, Plasmid Preparation, Sequencing

    20) Product Images from "Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells"

    Article Title: Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp640

    Targeted poly A trapping of the Oct4 locus. ( A ) Schematic representation of the targeted insertion of vectors pGTIV3 and pGTIV2 into the first intron of the mouse Oct4 locus. The location of the probe used for Southern blot analysis of the targeted clones is shown in red. The genomic organization of Oct4 is not drawn to scale. Unbiased insertional preference should theoretically give rise to neomycin resistant clones while tendency to insertion into the 3′ most intron should be associated with loss of neomycin resistance. ( B ) Top: number of G418 resistant colonies obtained after ES cell electroporation with the pGTIV3 and pGTIV2 Oct4 targeting vectors. The fractions of electroporated cells expressing Venus prior to G418 selection are indicated. Numbers and percentages are an average of three electroporation experiments. Bottom: Southern blot analysis of G418 resistant, Venus positive, pGTIV3 and pGTIV2- Oct4 targeted clones. Genomic DNA was digested using EcoRI (restriction sites are shown in A). Correctly targeted clones should yield an 11 kb (wild-type) and a 15 kb (targeted) band and are indicated by an asterisk. DNA from wild-type E14TG2a ES cells and an independently Oct4 targeted clone (expected bands of 6 and 11 kb) were also included as negative and positive controls, respectively (first two lanes from the left). The analysis of 15 pGTIV2- Oct4 targeted clones is also shown separately (bottom). ( C ) Targeted poly A trapping of Oct4 after promoter swap between vectors pGTIV3-Oct4 and pGTIV2-Oct4. The human β-actin promoter of the insertionally unbiased pGTIV3-Oct4 vector was exchanged for the PGK promoter present in the 3′ biased pGTIV2-Oct4 vector (represented by the arrow in A). Top: number of G418 resistant colonies obtained after electroporation with the pGTIV3-Oct4-PGK and pGTIV2-Oct4-β-actin modified constructs. Bottom: Southern blot analysis of G418 resistant, Venus positive clones electroporated with the pGTIV2-Oct4-β-actin vector. Genomic DNA was digested and probed as in B. Correctly targeted clones should yield an 11 kb (wild-type) and a 15 kb (targeted) band and are indicated by an asterisk; ( n = 2).
    Figure Legend Snippet: Targeted poly A trapping of the Oct4 locus. ( A ) Schematic representation of the targeted insertion of vectors pGTIV3 and pGTIV2 into the first intron of the mouse Oct4 locus. The location of the probe used for Southern blot analysis of the targeted clones is shown in red. The genomic organization of Oct4 is not drawn to scale. Unbiased insertional preference should theoretically give rise to neomycin resistant clones while tendency to insertion into the 3′ most intron should be associated with loss of neomycin resistance. ( B ) Top: number of G418 resistant colonies obtained after ES cell electroporation with the pGTIV3 and pGTIV2 Oct4 targeting vectors. The fractions of electroporated cells expressing Venus prior to G418 selection are indicated. Numbers and percentages are an average of three electroporation experiments. Bottom: Southern blot analysis of G418 resistant, Venus positive, pGTIV3 and pGTIV2- Oct4 targeted clones. Genomic DNA was digested using EcoRI (restriction sites are shown in A). Correctly targeted clones should yield an 11 kb (wild-type) and a 15 kb (targeted) band and are indicated by an asterisk. DNA from wild-type E14TG2a ES cells and an independently Oct4 targeted clone (expected bands of 6 and 11 kb) were also included as negative and positive controls, respectively (first two lanes from the left). The analysis of 15 pGTIV2- Oct4 targeted clones is also shown separately (bottom). ( C ) Targeted poly A trapping of Oct4 after promoter swap between vectors pGTIV3-Oct4 and pGTIV2-Oct4. The human β-actin promoter of the insertionally unbiased pGTIV3-Oct4 vector was exchanged for the PGK promoter present in the 3′ biased pGTIV2-Oct4 vector (represented by the arrow in A). Top: number of G418 resistant colonies obtained after electroporation with the pGTIV3-Oct4-PGK and pGTIV2-Oct4-β-actin modified constructs. Bottom: Southern blot analysis of G418 resistant, Venus positive clones electroporated with the pGTIV2-Oct4-β-actin vector. Genomic DNA was digested and probed as in B. Correctly targeted clones should yield an 11 kb (wild-type) and a 15 kb (targeted) band and are indicated by an asterisk; ( n = 2).

    Techniques Used: Southern Blot, Clone Assay, Electroporation, Expressing, Selection, Plasmid Preparation, Modification, Construct

    21) Product Images from "Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum"

    Article Title: Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum

    Journal: Molecular Biology International

    doi: 10.4061/2011/854626

    Effect of peptide WRWYCRCK on restriction digestion by restriction endonucleases. Representative gel picture showing the result of incubating pUC19 plasmid with EcoRI (lanes 4–7) or PvuII (lanes 8–11) in the presence of peptide WRWYCRCK at the following concentration: 12.5 μ M, 25 μ M, or 50 μ M. The sizes, kbp, of the DNA marker (lane 1, labeled M) are shown to the left of the gel picture. 0: control lanes with no peptide added; +: control lane with 50 μ M peptide added; asterisks indicate the gel electrophoretic mobility of the digested plasmid, for EcoRI, 2.7 kbp, and for PvuII, 0.3 kbp and 2.4 kbp.
    Figure Legend Snippet: Effect of peptide WRWYCRCK on restriction digestion by restriction endonucleases. Representative gel picture showing the result of incubating pUC19 plasmid with EcoRI (lanes 4–7) or PvuII (lanes 8–11) in the presence of peptide WRWYCRCK at the following concentration: 12.5 μ M, 25 μ M, or 50 μ M. The sizes, kbp, of the DNA marker (lane 1, labeled M) are shown to the left of the gel picture. 0: control lanes with no peptide added; +: control lane with 50 μ M peptide added; asterisks indicate the gel electrophoretic mobility of the digested plasmid, for EcoRI, 2.7 kbp, and for PvuII, 0.3 kbp and 2.4 kbp.

    Techniques Used: Plasmid Preparation, Concentration Assay, Marker, Labeling

    22) Product Images from "Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes"

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

    Journal: Journal of Virology

    doi: 10.1128/JVI.00736-17

    EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P
    Figure Legend Snippet: EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P

    Techniques Used: Expressing, DNA Methylation Assay, Binding Assay, Methylation, Negative Control, CpG Methylation Assay, Real-time Polymerase Chain Reaction

    23) Product Images from "CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection"

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.05.103

    CRISPR-READI optimization for efficient HDR editing in mouse embryos. a Zygotes were transduced with a panel of AAV serotypes harboring a CMV-eGFP reporter and imaged by fluorescent microscopy 48 hours post-transduction. Representative embryos transduced with scAAV1-CMV-eGFP are shown (left), and mean fluorescence intensity per embryo was quantified for each serotype (right). Scale bars = 50 μm. b Cartoon depiction of CRISPR-READI workflow. Embryos are collected from superovulated female mice, transduced with rAAV1 harboring the donor template, electroporated with Cas9/sgRNA RNPs, and implanted into pseudopregnant females to generate edited mice. c Schematic of Tyr targeting strategy. The scAAV1-Tyr donor creates an EcoRI restriction site in exon 1 of the Tyr locus upon HDR editing. ITR: inverted terminal repeat, HA: homology arm, F/R: forward/reverse primers for RFLP analysis. d Optimization of rAAV1 dosage for HDR editing. Zygotes were transduced with scAAV1-Tyr at a dose of 1.1×10 8 , 4.2×10 8 , or 1.7×10 9 GCs, electroporated with RNPs 5 hours post-transduction, and then returned to rAAV1 incubation for another 19 hours. Treated embryos were cultured to the morula stage and genotyped by restriction fragment length polymorphism (RFLP) analysis (shown for dose of 1.7×10 9 GCs). Edited embryos yield 650 bp and 420 bp bands upon EcoRI digestion of the PCR amplicon (top, black arrows). HDR rate was quantified by RFLP analysis for each dose (bottom left), and embryo viability was scored as percentage of cultured embryos that reached the morula stage (bottom right). e Optimization of RNP electroporation timing relative to rAAV transduction. Zygotes were transduced with scAAV1-Tyr, electroporated at varying time points post-transduction (2, 4, 6, 8, or 10 hours), and returned to rAAV incubation for a total of 24 hours. Treated embryos were cultured to the morula stage, lysed, and assessed by RFLP analysis (right). 6 hours (*) was identified as the optimal time of RNP electroporation for maximal editing efficiency.
    Figure Legend Snippet: CRISPR-READI optimization for efficient HDR editing in mouse embryos. a Zygotes were transduced with a panel of AAV serotypes harboring a CMV-eGFP reporter and imaged by fluorescent microscopy 48 hours post-transduction. Representative embryos transduced with scAAV1-CMV-eGFP are shown (left), and mean fluorescence intensity per embryo was quantified for each serotype (right). Scale bars = 50 μm. b Cartoon depiction of CRISPR-READI workflow. Embryos are collected from superovulated female mice, transduced with rAAV1 harboring the donor template, electroporated with Cas9/sgRNA RNPs, and implanted into pseudopregnant females to generate edited mice. c Schematic of Tyr targeting strategy. The scAAV1-Tyr donor creates an EcoRI restriction site in exon 1 of the Tyr locus upon HDR editing. ITR: inverted terminal repeat, HA: homology arm, F/R: forward/reverse primers for RFLP analysis. d Optimization of rAAV1 dosage for HDR editing. Zygotes were transduced with scAAV1-Tyr at a dose of 1.1×10 8 , 4.2×10 8 , or 1.7×10 9 GCs, electroporated with RNPs 5 hours post-transduction, and then returned to rAAV1 incubation for another 19 hours. Treated embryos were cultured to the morula stage and genotyped by restriction fragment length polymorphism (RFLP) analysis (shown for dose of 1.7×10 9 GCs). Edited embryos yield 650 bp and 420 bp bands upon EcoRI digestion of the PCR amplicon (top, black arrows). HDR rate was quantified by RFLP analysis for each dose (bottom left), and embryo viability was scored as percentage of cultured embryos that reached the morula stage (bottom right). e Optimization of RNP electroporation timing relative to rAAV transduction. Zygotes were transduced with scAAV1-Tyr, electroporated at varying time points post-transduction (2, 4, 6, 8, or 10 hours), and returned to rAAV incubation for a total of 24 hours. Treated embryos were cultured to the morula stage, lysed, and assessed by RFLP analysis (right). 6 hours (*) was identified as the optimal time of RNP electroporation for maximal editing efficiency.

    Techniques Used: CRISPR, Transduction, Microscopy, Fluorescence, Mouse Assay, Incubation, Cell Culture, Polymerase Chain Reaction, Amplification, Electroporation

    24) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    25) Product Images from "Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101"

    Article Title: Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101

    Journal: mBio

    doi: 10.1128/mBio.02674-18

    Characterization of the SM101-CPR1055KO null mutant and analysis of sporulation and CPE production. (A) PCR confirming insertional mutagenesis of th e cpr1055 gene in SM101-CPR1055. Shown is the cpr1055 PCR product amplified using DNA from wild-type SM101 (left lane) or the SM101-CPR1055KO mutant (right lane). Note that DNA from the null mutant strain supported amplification of a larger product due to the insertion of an intron into its cpr1055 gene. (B) Southern blot hybridization with an intron-specific probe with DNA from SM101 or SM101-CPR1055KO. The blot shows results of intron-specific Southern blot hybridization with DNA from wild-type SM101 (left lane) or the cpr1055 null mutant (middle lane). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the right lane is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected. However, a single intron-specific band was detected for the SM101-CPR1055KO mutant. (C) RT-PCR analysis for cpr1055 (top panel) or polC (middle panel) transcription in wild-type SM101 or the SM101-CPR1055KO mutant. SM101 DNA was used as a positive control (gDNA). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from both strains were free from DNA contamination, the samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101 versus the SM101-CPR1055KO mutant cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . (E) Comparison of results of sporulation by WT SM101 versus SM101-CPR1055KO. Both strains were grown overnight at 37°C in MDS and then subjected to heat shock treatment and plated on BHI agar. After overnight incubation in an anaerobic jar, the resultant colonies were counted and the counts were converted to numbers of spores per milliliter. (F) Comparison of levels of CPE production by SM101 versus the SM101-CPR1055KO mutant. Supernatants of WT SM101 or SM101-CPR1055KO were grown overnight at 37°C in MDS and then assessed by Western blotting for CPE. The results showed that CPE production remained strong after inactivation of the cpr1055 gene. All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.
    Figure Legend Snippet: Characterization of the SM101-CPR1055KO null mutant and analysis of sporulation and CPE production. (A) PCR confirming insertional mutagenesis of th e cpr1055 gene in SM101-CPR1055. Shown is the cpr1055 PCR product amplified using DNA from wild-type SM101 (left lane) or the SM101-CPR1055KO mutant (right lane). Note that DNA from the null mutant strain supported amplification of a larger product due to the insertion of an intron into its cpr1055 gene. (B) Southern blot hybridization with an intron-specific probe with DNA from SM101 or SM101-CPR1055KO. The blot shows results of intron-specific Southern blot hybridization with DNA from wild-type SM101 (left lane) or the cpr1055 null mutant (middle lane). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the right lane is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected. However, a single intron-specific band was detected for the SM101-CPR1055KO mutant. (C) RT-PCR analysis for cpr1055 (top panel) or polC (middle panel) transcription in wild-type SM101 or the SM101-CPR1055KO mutant. SM101 DNA was used as a positive control (gDNA). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from both strains were free from DNA contamination, the samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101 versus the SM101-CPR1055KO mutant cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . (E) Comparison of results of sporulation by WT SM101 versus SM101-CPR1055KO. Both strains were grown overnight at 37°C in MDS and then subjected to heat shock treatment and plated on BHI agar. After overnight incubation in an anaerobic jar, the resultant colonies were counted and the counts were converted to numbers of spores per milliliter. (F) Comparison of levels of CPE production by SM101 versus the SM101-CPR1055KO mutant. Supernatants of WT SM101 or SM101-CPR1055KO were grown overnight at 37°C in MDS and then assessed by Western blotting for CPE. The results showed that CPE production remained strong after inactivation of the cpr1055 gene. All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Cell Culture, Incubation, Western Blot

    Characterization of the SM101-CPR0195KO null mutant and SM101-CPR0195comp complementing strain. (A) PCR confirming insertional mutagenesis of the cpr0195 gene in SM101-0195KO. Shown is the cpr0195 PCR product amplified using DNA from wild-type SM101 (lane 2), the SM101-CPR0195KO mutant (lane 3), or the SM101-CPR0195comp complementing strain (lane 4). Note that, compared to the ∼300-bp product amplified using DNA containing a wild-type cpr0195 gene, DNA from the null mutant strain supported amplification of a larger (∼1,200-bp) product due to the insertion of an intron into its cpr0195 gene. (B) Southern blot hybridization of an intron-specific probe with DNA from SM101 (left), SM101-CPR0195KO (middle), or SM101-CPR0195comp (right). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the middle and right lanes is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected, while a single intron-specific band was detected for the SM101-CPR0195KO mutant and complementing strain. (C) RT-PCR analysis for cpr019 5 (top panel) or polC (middle panel) transcription in wild-type SM101, the SM101-CPR0195KO mutant, or the complementing strain. SM101 DNA was used as a positive control (gDNA [genomic DNA]). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from the three strains were free from DNA contamination, these samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101, the SM101-CPR0195KO mutant, and the SM101-CPR0195comp strain cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.
    Figure Legend Snippet: Characterization of the SM101-CPR0195KO null mutant and SM101-CPR0195comp complementing strain. (A) PCR confirming insertional mutagenesis of the cpr0195 gene in SM101-0195KO. Shown is the cpr0195 PCR product amplified using DNA from wild-type SM101 (lane 2), the SM101-CPR0195KO mutant (lane 3), or the SM101-CPR0195comp complementing strain (lane 4). Note that, compared to the ∼300-bp product amplified using DNA containing a wild-type cpr0195 gene, DNA from the null mutant strain supported amplification of a larger (∼1,200-bp) product due to the insertion of an intron into its cpr0195 gene. (B) Southern blot hybridization of an intron-specific probe with DNA from SM101 (left), SM101-CPR0195KO (middle), or SM101-CPR0195comp (right). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the middle and right lanes is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected, while a single intron-specific band was detected for the SM101-CPR0195KO mutant and complementing strain. (C) RT-PCR analysis for cpr019 5 (top panel) or polC (middle panel) transcription in wild-type SM101, the SM101-CPR0195KO mutant, or the complementing strain. SM101 DNA was used as a positive control (gDNA [genomic DNA]). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from the three strains were free from DNA contamination, these samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101, the SM101-CPR0195KO mutant, and the SM101-CPR0195comp strain cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Cell Culture

    26) Product Images from "Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo"

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

    Journal: Nature protocols

    doi: 10.1038/nprot.2015.094

    The parent vector and competitor vector used in this study. ( a ) Plasmid maps of the parent vector (i.e., pTGFP-T7-Hha10T) and the competitor vector (i.e., pTGFP-T7-Hha10comp). ( b ) Sequences of the parent and competitor vectors between the NheI and EcoRI
    Figure Legend Snippet: The parent vector and competitor vector used in this study. ( a ) Plasmid maps of the parent vector (i.e., pTGFP-T7-Hha10T) and the competitor vector (i.e., pTGFP-T7-Hha10comp). ( b ) Sequences of the parent and competitor vectors between the NheI and EcoRI

    Techniques Used: Plasmid Preparation

    27) Product Images from "Capsule Gene Analysis of Invasive Haemophilus influenzae: Accuracy of Serotyping and Prevalence of IS1016 among Nontypeable Isolates ▿"

    Article Title: Capsule Gene Analysis of Invasive Haemophilus influenzae: Accuracy of Serotyping and Prevalence of IS1016 among Nontypeable Isolates ▿

    Journal:

    doi: 10.1128/JCM.00794-07

    Southern hybridization of EcoRI-digested chromosomal DNA from Hib strain 1007 and Hib-minus strain GA346 probed with DIG-labeled pUO38. GA346 contains the major hybridizing bands corresponding to the Hib cap locus (20, 10.2, 4.4, 2.7, and 2.1 kb) and
    Figure Legend Snippet: Southern hybridization of EcoRI-digested chromosomal DNA from Hib strain 1007 and Hib-minus strain GA346 probed with DIG-labeled pUO38. GA346 contains the major hybridizing bands corresponding to the Hib cap locus (20, 10.2, 4.4, 2.7, and 2.1 kb) and

    Techniques Used: Hybridization, Labeling

    Southern hybridization analysis of representative isolates demonstrating hybridization with IS 1016 . Chromosomal DNA was digested with EcoRI from Hib 1007 (lane 2), Rd (lane 3), GA858 (lane 4), GA1354 (lane 5), GA4891 (lane 6), GA2078 (lane 7), GA3204
    Figure Legend Snippet: Southern hybridization analysis of representative isolates demonstrating hybridization with IS 1016 . Chromosomal DNA was digested with EcoRI from Hib 1007 (lane 2), Rd (lane 3), GA858 (lane 4), GA1354 (lane 5), GA4891 (lane 6), GA2078 (lane 7), GA3204

    Techniques Used: Hybridization

    28) Product Images from "Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells"

    Article Title: Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells

    Journal: Radiation Oncology (London, England)

    doi: 10.1186/s13014-017-0941-6

    TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c - d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown
    Figure Legend Snippet: TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c - d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown

    Techniques Used: Homologous Recombination, Transfection, Plasmid Preparation, Activity Assay, Irradiation, Immunofluorescence, Staining

    29) Product Images from "Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles"

    Article Title: Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx427

    Circular permutation polymerase chain reaction (PCR) strategy used for preparing nucleosomal DNA with T 11 -tracts at specific superhelix locations (SHLs). ( A ) A 168-mer DNA duplex was designed to have a centrally located T 11 -tract (underlined), terminal EcoRI restriction sites (green italic), phased minor groove bending (T/A) 3 sequences in red and major groove bending (G/C) 3 sequences in blue. The T in orange in the T 11 -tract corresponds to the position at which the major groove is expected to bend toward the histone surface at the dyad axis. ( B ) The 168-mer DNA duplex was prepared by primer extension of two overlapping 95 and 96-mers, cleaved with EcoRI and multimerized with T4 DNA ligase and adenosine triphosphate. The dimer was then excised from a gel and cloned. Nucleosomal DNAs with T 11 -tracts at specific SHLs were prepared from the clone by PCR using specific pairs of forward and reverse primers ( Supplementary Figure S3 ) whose positions are shown as solid and dashed arrows respectively on the sequence in panel A. The SHLs are identified by the number of helical turns from the dyad axis on the blue strand containing the T 11 -tract. They are negative to correspond with the negative numbers assigned to nucleotides on the T 11 -tract strand that are 5΄-to the T at the dyad axis which is assigned as 0. The −3 (inside) and +3 (outside) nucleotide positions are shown in blue. The positions at which the major and minor grooves face the histone surface are indicated by M and m, respectively, and are colored coded blue and red to match the major groove and minor groove bending motifs in panel A.
    Figure Legend Snippet: Circular permutation polymerase chain reaction (PCR) strategy used for preparing nucleosomal DNA with T 11 -tracts at specific superhelix locations (SHLs). ( A ) A 168-mer DNA duplex was designed to have a centrally located T 11 -tract (underlined), terminal EcoRI restriction sites (green italic), phased minor groove bending (T/A) 3 sequences in red and major groove bending (G/C) 3 sequences in blue. The T in orange in the T 11 -tract corresponds to the position at which the major groove is expected to bend toward the histone surface at the dyad axis. ( B ) The 168-mer DNA duplex was prepared by primer extension of two overlapping 95 and 96-mers, cleaved with EcoRI and multimerized with T4 DNA ligase and adenosine triphosphate. The dimer was then excised from a gel and cloned. Nucleosomal DNAs with T 11 -tracts at specific SHLs were prepared from the clone by PCR using specific pairs of forward and reverse primers ( Supplementary Figure S3 ) whose positions are shown as solid and dashed arrows respectively on the sequence in panel A. The SHLs are identified by the number of helical turns from the dyad axis on the blue strand containing the T 11 -tract. They are negative to correspond with the negative numbers assigned to nucleotides on the T 11 -tract strand that are 5΄-to the T at the dyad axis which is assigned as 0. The −3 (inside) and +3 (outside) nucleotide positions are shown in blue. The positions at which the major and minor grooves face the histone surface are indicated by M and m, respectively, and are colored coded blue and red to match the major groove and minor groove bending motifs in panel A.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Sequencing

    30) Product Images from "TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression"

    Article Title: TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186568

    pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.
    Figure Legend Snippet: pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.

    Techniques Used: Positron Emission Tomography

    The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.
    Figure Legend Snippet: The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.

    Techniques Used: Expressing, Plasmid Preparation, Positron Emission Tomography, Derivative Assay, Selection, Transformation Assay, Clone Assay, Recombinant, Ligation

    31) Product Images from "Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101"

    Article Title: Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101

    Journal: mBio

    doi: 10.1128/mBio.02674-18

    Characterization of the SM101-CPR1055KO null mutant and analysis of sporulation and CPE production. (A) PCR confirming insertional mutagenesis of th e cpr1055 gene in SM101-CPR1055. Shown is the cpr1055 PCR product amplified using DNA from wild-type SM101 (left lane) or the SM101-CPR1055KO mutant (right lane). Note that DNA from the null mutant strain supported amplification of a larger product due to the insertion of an intron into its cpr1055 gene. (B) Southern blot hybridization with an intron-specific probe with DNA from SM101 or SM101-CPR1055KO. The blot shows results of intron-specific Southern blot hybridization with DNA from wild-type SM101 (left lane) or the cpr1055 null mutant (middle lane). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the right lane is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected. However, a single intron-specific band was detected for the SM101-CPR1055KO mutant. (C) RT-PCR analysis for cpr1055 (top panel) or polC (middle panel) transcription in wild-type SM101 or the SM101-CPR1055KO mutant. SM101 DNA was used as a positive control (gDNA). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from both strains were free from DNA contamination, the samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101 versus the SM101-CPR1055KO mutant cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . (E) Comparison of results of sporulation by WT SM101 versus SM101-CPR1055KO. Both strains were grown overnight at 37°C in MDS and then subjected to heat shock treatment and plated on BHI agar. After overnight incubation in an anaerobic jar, the resultant colonies were counted and the counts were converted to numbers of spores per milliliter. (F) Comparison of levels of CPE production by SM101 versus the SM101-CPR1055KO mutant. Supernatants of WT SM101 or SM101-CPR1055KO were grown overnight at 37°C in MDS and then assessed by Western blotting for CPE. The results showed that CPE production remained strong after inactivation of the cpr1055 gene. All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.
    Figure Legend Snippet: Characterization of the SM101-CPR1055KO null mutant and analysis of sporulation and CPE production. (A) PCR confirming insertional mutagenesis of th e cpr1055 gene in SM101-CPR1055. Shown is the cpr1055 PCR product amplified using DNA from wild-type SM101 (left lane) or the SM101-CPR1055KO mutant (right lane). Note that DNA from the null mutant strain supported amplification of a larger product due to the insertion of an intron into its cpr1055 gene. (B) Southern blot hybridization with an intron-specific probe with DNA from SM101 or SM101-CPR1055KO. The blot shows results of intron-specific Southern blot hybridization with DNA from wild-type SM101 (left lane) or the cpr1055 null mutant (middle lane). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the right lane is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected. However, a single intron-specific band was detected for the SM101-CPR1055KO mutant. (C) RT-PCR analysis for cpr1055 (top panel) or polC (middle panel) transcription in wild-type SM101 or the SM101-CPR1055KO mutant. SM101 DNA was used as a positive control (gDNA). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from both strains were free from DNA contamination, the samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101 versus the SM101-CPR1055KO mutant cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . (E) Comparison of results of sporulation by WT SM101 versus SM101-CPR1055KO. Both strains were grown overnight at 37°C in MDS and then subjected to heat shock treatment and plated on BHI agar. After overnight incubation in an anaerobic jar, the resultant colonies were counted and the counts were converted to numbers of spores per milliliter. (F) Comparison of levels of CPE production by SM101 versus the SM101-CPR1055KO mutant. Supernatants of WT SM101 or SM101-CPR1055KO were grown overnight at 37°C in MDS and then assessed by Western blotting for CPE. The results showed that CPE production remained strong after inactivation of the cpr1055 gene. All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Cell Culture, Incubation, Western Blot

    Characterization of the SM101-CPR0195KO null mutant and SM101-CPR0195comp complementing strain. (A) PCR confirming insertional mutagenesis of the cpr0195 gene in SM101-0195KO. Shown is the cpr0195 PCR product amplified using DNA from wild-type SM101 (lane 2), the SM101-CPR0195KO mutant (lane 3), or the SM101-CPR0195comp complementing strain (lane 4). Note that, compared to the ∼300-bp product amplified using DNA containing a wild-type cpr0195 gene, DNA from the null mutant strain supported amplification of a larger (∼1,200-bp) product due to the insertion of an intron into its cpr0195 gene. (B) Southern blot hybridization of an intron-specific probe with DNA from SM101 (left), SM101-CPR0195KO (middle), or SM101-CPR0195comp (right). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the middle and right lanes is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected, while a single intron-specific band was detected for the SM101-CPR0195KO mutant and complementing strain. (C) RT-PCR analysis for cpr019 5 (top panel) or polC (middle panel) transcription in wild-type SM101, the SM101-CPR0195KO mutant, or the complementing strain. SM101 DNA was used as a positive control (gDNA [genomic DNA]). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from the three strains were free from DNA contamination, these samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101, the SM101-CPR0195KO mutant, and the SM101-CPR0195comp strain cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.
    Figure Legend Snippet: Characterization of the SM101-CPR0195KO null mutant and SM101-CPR0195comp complementing strain. (A) PCR confirming insertional mutagenesis of the cpr0195 gene in SM101-0195KO. Shown is the cpr0195 PCR product amplified using DNA from wild-type SM101 (lane 2), the SM101-CPR0195KO mutant (lane 3), or the SM101-CPR0195comp complementing strain (lane 4). Note that, compared to the ∼300-bp product amplified using DNA containing a wild-type cpr0195 gene, DNA from the null mutant strain supported amplification of a larger (∼1,200-bp) product due to the insertion of an intron into its cpr0195 gene. (B) Southern blot hybridization of an intron-specific probe with DNA from SM101 (left), SM101-CPR0195KO (middle), or SM101-CPR0195comp (right). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the middle and right lanes is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected, while a single intron-specific band was detected for the SM101-CPR0195KO mutant and complementing strain. (C) RT-PCR analysis for cpr019 5 (top panel) or polC (middle panel) transcription in wild-type SM101, the SM101-CPR0195KO mutant, or the complementing strain. SM101 DNA was used as a positive control (gDNA [genomic DNA]). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from the three strains were free from DNA contamination, these samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101, the SM101-CPR0195KO mutant, and the SM101-CPR0195comp strain cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Cell Culture

    32) Product Images from "A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells"

    Article Title: A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066084

    Intracellular uptake of peptides and anti-Ras scFv fusion proteins and their anti-proliferative activity. (A) Schematic representation of peptide-anti-Ras scFv cDNA constructs. DNA encoding peptides and the Y13-259-scFv cDNA were fused as described in Materials and Methods and cloned into the Nco I and EcoR I sites of pET21c. The white boxes represent V H and V L of the Y13-259 scFv sequence. The round box indicates the sequence encoding the peptides: Tat or BR2. The Nco I and EcoR I restriction sites and stop codon positions are also indicated. (B) Protein uptake was analyzed by Western blotting of fractionated lysates from HCT116 cells treated with PBS, anti-Ras scFv, BR2- or Tat-scFv fusion protein (each, 2 µM) at 37°C for 2 h. An anti-His antibody was used to detect intracellular Tat-scFv and BR2-scFv (28-kDa). (C) The anti-proliferative activity of peptides and anti-Ras scFv fusion proteins. HCT116 cells were exposed to the indicated concentrations of anti-Ras scFv, Tat- or BR2-scFv fusion protein at 37°C for 24 h. Cell proliferation was determined using the MTT assay. Data represent the mean ± s.d. of three independent experiments.
    Figure Legend Snippet: Intracellular uptake of peptides and anti-Ras scFv fusion proteins and their anti-proliferative activity. (A) Schematic representation of peptide-anti-Ras scFv cDNA constructs. DNA encoding peptides and the Y13-259-scFv cDNA were fused as described in Materials and Methods and cloned into the Nco I and EcoR I sites of pET21c. The white boxes represent V H and V L of the Y13-259 scFv sequence. The round box indicates the sequence encoding the peptides: Tat or BR2. The Nco I and EcoR I restriction sites and stop codon positions are also indicated. (B) Protein uptake was analyzed by Western blotting of fractionated lysates from HCT116 cells treated with PBS, anti-Ras scFv, BR2- or Tat-scFv fusion protein (each, 2 µM) at 37°C for 2 h. An anti-His antibody was used to detect intracellular Tat-scFv and BR2-scFv (28-kDa). (C) The anti-proliferative activity of peptides and anti-Ras scFv fusion proteins. HCT116 cells were exposed to the indicated concentrations of anti-Ras scFv, Tat- or BR2-scFv fusion protein at 37°C for 24 h. Cell proliferation was determined using the MTT assay. Data represent the mean ± s.d. of three independent experiments.

    Techniques Used: Activity Assay, Construct, Clone Assay, Sequencing, Western Blot, MTT Assay

    33) Product Images from "Detection of Molecular Diversity in Bacillus atrophaeus by Amplified Fragment Length Polymorphism Analysis"

    Article Title: Detection of Molecular Diversity in Bacillus atrophaeus by Amplified Fragment Length Polymorphism Analysis

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.5.2786-2790.2004

    Digitized AFLP patterns of Bacillus taxa generated using primer sets EcoRI plus C/MseI plus CA (A) and EcoRI plus C/MseI plus CC (B). Across the top of each image is the fragment size scale (in bases). The Bacillus species and strain designations for
    Figure Legend Snippet: Digitized AFLP patterns of Bacillus taxa generated using primer sets EcoRI plus C/MseI plus CA (A) and EcoRI plus C/MseI plus CC (B). Across the top of each image is the fragment size scale (in bases). The Bacillus species and strain designations for

    Techniques Used: Generated

    34) Product Images from "Polymorphic Integrations of an Endogenous Gammaretrovirus in the Mule Deer Genome"

    Article Title: Polymorphic Integrations of an Endogenous Gammaretrovirus in the Mule Deer Genome

    Journal: Journal of Virology

    doi: 10.1128/JVI.06859-11

    Southern blot analysis of CrERVγ integrations in the mule deer genome. Mule deer genomic DNAs digested with EcoRI and transferred to a membrane were hybridized with a CrERVγ gag-pro probe to reveal the 5′ virus-host junction fragments
    Figure Legend Snippet: Southern blot analysis of CrERVγ integrations in the mule deer genome. Mule deer genomic DNAs digested with EcoRI and transferred to a membrane were hybridized with a CrERVγ gag-pro probe to reveal the 5′ virus-host junction fragments

    Techniques Used: Southern Blot

    35) Product Images from "Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio"

    Article Title: Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio

    Journal: Journal of aquatic animal health

    doi: 10.1080/08997659.2013.782226

    Linearized plasmid profiles of Edwardsiella ictaluri isolates. Plasmid DNA from E. ictaluri isolated from Channel Catfish or Zebrafish was digested with EcoRI or BstZ17I, respectively, and separated by 0.6% agarose gel electrophoresis using a 1-kb DNA
    Figure Legend Snippet: Linearized plasmid profiles of Edwardsiella ictaluri isolates. Plasmid DNA from E. ictaluri isolated from Channel Catfish or Zebrafish was digested with EcoRI or BstZ17I, respectively, and separated by 0.6% agarose gel electrophoresis using a 1-kb DNA

    Techniques Used: Plasmid Preparation, Isolation, Agarose Gel Electrophoresis

    36) Product Images from "Paired cloning of the T cell receptor ? and ? genes from a single T cell without the establishment of a T cell clone"

    Article Title: Paired cloning of the T cell receptor ? and ? genes from a single T cell without the establishment of a T cell clone

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2001.01437.x

    Cloning and expression of the TCR scFv. TCR α genes of Vα12 + T cells and TCR β genes of the two clonally expanded T cells (A22 and P39) were cloned for single strand conformation polymorphism (SSCP), nucleotide sequencing and/or expressing the TCR scFv protein. (a) Location of each primer is indicated by arrows. SS, Signal sequence. (b) The TCR genes of A22 were subcloned to plasmid vectors for expressing the scFv protein. The TCR α and β genes were subcloned to V L and V H sites of 9F12-encoding pCANTAB5E, respectively. The entire gene of TCR α/linker/TCRβ/E-Tag was transferred to EcoRI/BamHI-digested pMAL-c vector. The TCR scFv was expressed as a fusion protein with maltose binding protein (MBP) and E-Tag. After the MBP was cleaved by Factor Xa, the TCR scFv/E-Tag was purified on an affinity column using the anti-E-Tag antibody.
    Figure Legend Snippet: Cloning and expression of the TCR scFv. TCR α genes of Vα12 + T cells and TCR β genes of the two clonally expanded T cells (A22 and P39) were cloned for single strand conformation polymorphism (SSCP), nucleotide sequencing and/or expressing the TCR scFv protein. (a) Location of each primer is indicated by arrows. SS, Signal sequence. (b) The TCR genes of A22 were subcloned to plasmid vectors for expressing the scFv protein. The TCR α and β genes were subcloned to V L and V H sites of 9F12-encoding pCANTAB5E, respectively. The entire gene of TCR α/linker/TCRβ/E-Tag was transferred to EcoRI/BamHI-digested pMAL-c vector. The TCR scFv was expressed as a fusion protein with maltose binding protein (MBP) and E-Tag. After the MBP was cleaved by Factor Xa, the TCR scFv/E-Tag was purified on an affinity column using the anti-E-Tag antibody.

    Techniques Used: Clone Assay, Expressing, Sequencing, Plasmid Preparation, Binding Assay, Purification, Affinity Column

    37) Product Images from "Activation of XerCD-dif recombination by the FtsK DNA translocase"

    Article Title: Activation of XerCD-dif recombination by the FtsK DNA translocase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr078

    ( A ) Recombination in the presence of peptide WRWYCR traps HJs. Recombination was carried out in the presence of the indicated concentration of peptide and subsequently cut with EcoRI so that HJs migrate slowly. ( B) Denaturing alkali gels allow determination of exchanged strands in isolated HJs. Isolated HJs were 5′-end labelled at each EcoRI cut site. Subsequently, some of the DNA was then further digested with ScaI, and samples were then denatured and electrophoresed. The relative positions of each site are shown diagrammatically below the gel. Sizes of the four strands resulting from EcoRI digestion are shown alongside (left). The expected sizes of top strand exchange (XerC-mediated) and bottom strand exchange (XerD-mediated) are shown on the right. Two strand sizes (3038 and 780) are specific for XerD mediated exchange (shown in bold), while XerC mediated exchange produces two different diagnostic product sizes (2212 and 1613, also in bold). The other strand sizes (415 and 1727) are common to both events. Note that there is always a background of XerC-mediated exchange, which can be estimated from the CD alone lane. However, upon stimulation by γ (in any form) the level of XerD-mediated exchange is greatly increased. Note that there was partial digestion by ScaI of the XerCD + γ 3 reaction (far right lane) so that the four bands seen with EcoRI digestion are still present.
    Figure Legend Snippet: ( A ) Recombination in the presence of peptide WRWYCR traps HJs. Recombination was carried out in the presence of the indicated concentration of peptide and subsequently cut with EcoRI so that HJs migrate slowly. ( B) Denaturing alkali gels allow determination of exchanged strands in isolated HJs. Isolated HJs were 5′-end labelled at each EcoRI cut site. Subsequently, some of the DNA was then further digested with ScaI, and samples were then denatured and electrophoresed. The relative positions of each site are shown diagrammatically below the gel. Sizes of the four strands resulting from EcoRI digestion are shown alongside (left). The expected sizes of top strand exchange (XerC-mediated) and bottom strand exchange (XerD-mediated) are shown on the right. Two strand sizes (3038 and 780) are specific for XerD mediated exchange (shown in bold), while XerC mediated exchange produces two different diagnostic product sizes (2212 and 1613, also in bold). The other strand sizes (415 and 1727) are common to both events. Note that there is always a background of XerC-mediated exchange, which can be estimated from the CD alone lane. However, upon stimulation by γ (in any form) the level of XerD-mediated exchange is greatly increased. Note that there was partial digestion by ScaI of the XerCD + γ 3 reaction (far right lane) so that the four bands seen with EcoRI digestion are still present.

    Techniques Used: Concentration Assay, Isolation, Diagnostic Assay

    ( A ) Schematic of recombination reactions; FtsK dependent recombination gives exclusively free products (P1 + P2) whereas the XerCγ or XerDγ fusion proteins produce mainly catenated products with a small amount of free P1 + P2. Catenated products up to six crossings (6-cat) are shown but higher forms are apparent in the gels. Second recombination events on the catenanes can produce knotted products, both twist and torus knots depending upon the synaptic complexity. DNA supercoiling is not shown for clarity. ( B ) Recombination reactions (20 min) with the indicated proteins were nicked with DNaseI in the presence of ethidium, to reveal the presence of catenated/knotted recombination products. Both fusion proteins produce catenanes, whereas FtsK (K) with XerCD (CD) does not. Nicking was not complete leaving some supercoiled plasmid substrate (supercoiled S). The smaller product, P2, was present lower down the gel but is not shown here. ( C ) Timecourse of recombination with XerCγD proteins. Reactions were nicked as previously. Catenanes (as labelled; the 10-crossing catenane co-migrates with nicked P1) appear at early time points and become stronger upon incubation, concomitant with the appearance of knotted products (weaker unlabelled bands, interdigitated with the catenanes). ( D ) Recombination reactions with the indicated proteins were either digested with EcoRI to show clearly the amount of recombination (upper panel) or electrophoresed without cutting (lower panel) to show the level of free circle product. Supercoiled catenated products were not resolved from the supercoiled substrate in the lower gel.
    Figure Legend Snippet: ( A ) Schematic of recombination reactions; FtsK dependent recombination gives exclusively free products (P1 + P2) whereas the XerCγ or XerDγ fusion proteins produce mainly catenated products with a small amount of free P1 + P2. Catenated products up to six crossings (6-cat) are shown but higher forms are apparent in the gels. Second recombination events on the catenanes can produce knotted products, both twist and torus knots depending upon the synaptic complexity. DNA supercoiling is not shown for clarity. ( B ) Recombination reactions (20 min) with the indicated proteins were nicked with DNaseI in the presence of ethidium, to reveal the presence of catenated/knotted recombination products. Both fusion proteins produce catenanes, whereas FtsK (K) with XerCD (CD) does not. Nicking was not complete leaving some supercoiled plasmid substrate (supercoiled S). The smaller product, P2, was present lower down the gel but is not shown here. ( C ) Timecourse of recombination with XerCγD proteins. Reactions were nicked as previously. Catenanes (as labelled; the 10-crossing catenane co-migrates with nicked P1) appear at early time points and become stronger upon incubation, concomitant with the appearance of knotted products (weaker unlabelled bands, interdigitated with the catenanes). ( D ) Recombination reactions with the indicated proteins were either digested with EcoRI to show clearly the amount of recombination (upper panel) or electrophoresed without cutting (lower panel) to show the level of free circle product. Supercoiled catenated products were not resolved from the supercoiled substrate in the lower gel.

    Techniques Used: Plasmid Preparation, Incubation

    38) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.
    Figure Legend Snippet: Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.

    Techniques Used: Inhibition

    ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.
    Figure Legend Snippet: ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.

    Techniques Used: Generated, Incubation

    39) Product Images from "NanR Regulates Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain F4969"

    Article Title: NanR Regulates Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain F4969

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00416-18

    (A) Preparation and characterization of a nanI and nanR double null mutant strain. The left lane shows a 1-kb molecular ruler (Thermo Fisher). The second and third lanes show the nanI PCR product amplified using DNA from wild-type strain F4969 or the nanI nanR double null mutant strain. The fifth and sixth lanes show the nanR PCR product amplified using DNA from wild-type strain F4969 or the nanI nanR double null mutant. Note that DNA from the double null mutant strain supported amplification of larger nanR and nanI products due to the insertion of an intron into the nanI and nanR genes of the double mutant. (B) Intron-specific Southern blot hybridization with DNA from wild-type F4969, single nanI and nanR mutants, or the double null mutant strain. DNA from each strain was digested with EcoRI overnight at 37°C and electrophoresed on a 1% agarose gel. The sizes of DNA fragments are shown to the left. Using DNA from wild-type F4969, no intron-specific band was detected. However, a single intron-specific band was detected for the nanI or nanR null mutant strains, while two intron-specific bands were detected for the double null mutant strain. (C) RT-PCR analysis for 16S RNA (top), nanI (middle), or nanR (bottom) transcription of wild-type F4969, the double null mutant (F4969DKO), and reversed double null mutant strain (F4969DKOrev). Wild-type F4969 DNA was used as a positive control. The leftmost, unlabeled lane contains a 1-kb molecular ruler (Thermo Fisher).
    Figure Legend Snippet: (A) Preparation and characterization of a nanI and nanR double null mutant strain. The left lane shows a 1-kb molecular ruler (Thermo Fisher). The second and third lanes show the nanI PCR product amplified using DNA from wild-type strain F4969 or the nanI nanR double null mutant strain. The fifth and sixth lanes show the nanR PCR product amplified using DNA from wild-type strain F4969 or the nanI nanR double null mutant. Note that DNA from the double null mutant strain supported amplification of larger nanR and nanI products due to the insertion of an intron into the nanI and nanR genes of the double mutant. (B) Intron-specific Southern blot hybridization with DNA from wild-type F4969, single nanI and nanR mutants, or the double null mutant strain. DNA from each strain was digested with EcoRI overnight at 37°C and electrophoresed on a 1% agarose gel. The sizes of DNA fragments are shown to the left. Using DNA from wild-type F4969, no intron-specific band was detected. However, a single intron-specific band was detected for the nanI or nanR null mutant strains, while two intron-specific bands were detected for the double null mutant strain. (C) RT-PCR analysis for 16S RNA (top), nanI (middle), or nanR (bottom) transcription of wild-type F4969, the double null mutant (F4969DKO), and reversed double null mutant strain (F4969DKOrev). Wild-type F4969 DNA was used as a positive control. The leftmost, unlabeled lane contains a 1-kb molecular ruler (Thermo Fisher).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control

    40) Product Images from "Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases"

    Article Title: Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0494-2

    Schematic overview of the OCT4-EI-TALEN construction and the SSA assay for testing the OCT4-TALEN-coding plasmids. a The TALEN-targeted sequences within exon 1 of the OCT4 gene are labeled in red. Assembled Repeat Variable Diresidue (RVD) repeats are represented schematically as boxes and labeled in yellow, red, blue and green. b The overview of the GFP-reporter system for SSA. Red lines represent 541 bp oligonucleotides near the TALEN-targeted sites amplified from genomic DNA. c Fluorescence intensity of GFP in 293 T cells 19 h or 49 h post transfection with OCT4-E1-TALEN-coding plasmids and the reporter vector pJL4-SSA (left) or controls transfected only with the reporter plasmid (right)
    Figure Legend Snippet: Schematic overview of the OCT4-EI-TALEN construction and the SSA assay for testing the OCT4-TALEN-coding plasmids. a The TALEN-targeted sequences within exon 1 of the OCT4 gene are labeled in red. Assembled Repeat Variable Diresidue (RVD) repeats are represented schematically as boxes and labeled in yellow, red, blue and green. b The overview of the GFP-reporter system for SSA. Red lines represent 541 bp oligonucleotides near the TALEN-targeted sites amplified from genomic DNA. c Fluorescence intensity of GFP in 293 T cells 19 h or 49 h post transfection with OCT4-E1-TALEN-coding plasmids and the reporter vector pJL4-SSA (left) or controls transfected only with the reporter plasmid (right)

    Techniques Used: SSA Assay, Labeling, Amplification, Fluorescence, Transfection, Plasmid Preparation

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    Article Title: Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker
    Article Snippet: .. The products were then cloned into vector pZIPPY-neo/gus [ ], which had been linearized with EcoRI and NotI (New England Biolabs, Ipswich, MA, USA) to generate the novel transfer vector pSPV-EGFP (Figure ). .. The plasmid was propagated in Escherichia coli strain Top10 (Invitrogen, Carlsbad, CA, USA).

    Amplification:

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: .. Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. ..

    Modification:

    Article Title: Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli
    Article Snippet: .. Construction of modified pB42 vectors, and preparation for ligation to insert DNA Plasmid pB42 was digested with XhoI and EcoRI to completion, and precipitated and digested with CIP (NEB). .. This vector was then split into three reactions, where three pairs of oligos were added to form the new multiple cloning sites.

    Ligation:

    Article Title: Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli
    Article Snippet: .. Construction of modified pB42 vectors, and preparation for ligation to insert DNA Plasmid pB42 was digested with XhoI and EcoRI to completion, and precipitated and digested with CIP (NEB). .. This vector was then split into three reactions, where three pairs of oligos were added to form the new multiple cloning sites.

    Polymerase Chain Reaction:

    Article Title: Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates
    Article Snippet: .. EcoRI, Eco91l, HindIII and PstI, for complete digestion with the recommended RE buffers in separate PCR tubes in the following reaction volume: 7 µl nuclease-free water, 10 µl DNA, 2 µl 10× RE buffer, 1 µl (10 U) EcoRI (New England Biolabs)/1 µl (10 U) Eco91l (Fermentas) / 1 µl (10 U) HindIII (Fermentas) / 1 µl (10 U) PstI (Fermentas). ..

    Incubation:

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: .. Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. ..

    Sequencing:

    Article Title: Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon
    Article Snippet: .. The authenticity of the plasmid construction was verified by EcoRI, AgeI and AscI (New England Biolabs) digestion (Fig. ) and by sequencing as previously described [ ]. .. This information indicated that eight substitutions were present in the RC coding region compared to the nucleotide sequence of passage 20 of the parental strain SAVH20/03 (Table ).

    Plasmid Preparation:

    Article Title: Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences
    Article Snippet: .. The colonies were picked into 10 mL LB with 50 μg/mL apramycin, and the plasmid DNA was extracted using a Miniprep kit (Qiagen, Valencia, CA, USA) and screened with restriction enzymes EcoRI and AvaI for pJGW92 and NcoI and AvaI for pJGW93 (NEB). .. The National Center for Biotechnology Information (NCBI) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to search for homologous proteins to known RecA, LexA, and DNA Pol V in the Clostridium thermocellum DSM 1313 genome.

    Article Title: Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli
    Article Snippet: .. Construction of modified pB42 vectors, and preparation for ligation to insert DNA Plasmid pB42 was digested with XhoI and EcoRI to completion, and precipitated and digested with CIP (NEB). .. This vector was then split into three reactions, where three pairs of oligos were added to form the new multiple cloning sites.

    Article Title: Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker
    Article Snippet: .. The products were then cloned into vector pZIPPY-neo/gus [ ], which had been linearized with EcoRI and NotI (New England Biolabs, Ipswich, MA, USA) to generate the novel transfer vector pSPV-EGFP (Figure ). .. The plasmid was propagated in Escherichia coli strain Top10 (Invitrogen, Carlsbad, CA, USA).

    Article Title: Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon
    Article Snippet: .. The authenticity of the plasmid construction was verified by EcoRI, AgeI and AscI (New England Biolabs) digestion (Fig. ) and by sequencing as previously described [ ]. .. This information indicated that eight substitutions were present in the RC coding region compared to the nucleotide sequence of passage 20 of the parental strain SAVH20/03 (Table ).

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    New England Biolabs restriction enzymes ecori
    The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with <t>AvaI</t> and <t>EcoRI.</t> The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown
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    The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Journal: Journal of industrial microbiology & biotechnology

    Article Title: Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

    doi: 10.1007/s10295-018-2049-x

    Figure Lengend Snippet: The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Article Snippet: The colonies were picked into 10 mL LB with 50 μg/mL apramycin, and the plasmid DNA was extracted using a Miniprep kit (Qiagen, Valencia, CA, USA) and screened with restriction enzymes EcoRI and AvaI for pJGW92 and NcoI and AvaI for pJGW93 (NEB).

    Techniques: Plasmid Preparation, Derivative Assay, Purification, Isolation, Transformation Assay, Polymerase Chain Reaction, Molecular Weight

    Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Journal: PLoS ONE

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    doi: 10.1371/journal.pone.0106713

    Figure Lengend Snippet: Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Article Snippet: We then double-digest ∼1.0 µg of DNA with high fidelity versions of the SbfI and EcoRI restriction enzymes (New England Biolabs); when less DNA is available, we have had good success starting with as little as 0.17 µg of genomic DNA.

    Techniques: Sequencing

    Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Journal: PLoS ONE

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    doi: 10.1371/journal.pone.0106713

    Figure Lengend Snippet: Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Article Snippet: We then double-digest ∼1.0 µg of DNA with high fidelity versions of the SbfI and EcoRI restriction enzymes (New England Biolabs); when less DNA is available, we have had good success starting with as little as 0.17 µg of genomic DNA.

    Techniques: Sequencing, Isolation, Agarose Gel Electrophoresis

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1.

    Techniques: Southern Blot, Incubation, Generated

    RE digestion of TE-PCR product with EcoRI, Pst I, Eco91l and HindIII . [ Lane M 100 bp DNA ladder; Lane 1 TE-PCR product with out RE; Lane 2 TE-PCR product with EcoRI ; Lane 3 TE-PCR product with Pst I ; Lane 5 TE-PCR product with out RE; Lane 6

    Journal: Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology

    Article Title: Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates

    doi: 10.1007/s12639-014-0582-8

    Figure Lengend Snippet: RE digestion of TE-PCR product with EcoRI, Pst I, Eco91l and HindIII . [ Lane M 100 bp DNA ladder; Lane 1 TE-PCR product with out RE; Lane 2 TE-PCR product with EcoRI ; Lane 3 TE-PCR product with Pst I ; Lane 5 TE-PCR product with out RE; Lane 6

    Article Snippet: EcoRI, Eco91l, HindIII and PstI, for complete digestion with the recommended RE buffers in separate PCR tubes in the following reaction volume: 7 µl nuclease-free water, 10 µl DNA, 2 µl 10× RE buffer, 1 µl (10 U) EcoRI (New England Biolabs)/1 µl (10 U) Eco91l (Fermentas) / 1 µl (10 U) HindIII (Fermentas) / 1 µl (10 U) PstI (Fermentas).

    Techniques: Polymerase Chain Reaction