ecori  (New England Biolabs)


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    Structured Review

    New England Biolabs ecori
    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 <t>NheI</t> and <t>EcoRI</t>
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    Images

    1) 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

    2) Product Images from "Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies"

    Article Title: Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

    Journal: bioRxiv

    doi: 10.1101/2021.04.16.440240

    Endogenous VHH05- or 127D01-tagging using CRISPR/Cas9. Workflow ( A ), fly embryos transformation ( B ), genotyping ( C ), and applications ( D ). ( A’ ) Common scarless vectors used for constructing donors. N- terminal vectors (PHD-N-3x127D01-donor or PHD-N-3xVHH05-donor) and C-terminal vectors (PHD-C-3x127D01-donor or PHD-C-3xVHH05-donor) contain EcoRI restriction enzyme sites that introduce the homologous arm sequences into donors. ( B’ ) Workflow example for introducing knock-in (KI) tags into the third chromosome. ( C’ ) Genotyping example of KI 3xVHH05 and 3x127D01 into the C-terminus of H2Av. Gel results showing the 5’ and 3’ PCR junctions. Representative sequencing chromatogram of PCR products from the junction PCR. ( D’ ) Immunostaining of H2Av- 3x127D01 and H2Av-3xVHH05. Adult male or female guts were dissected and stained with Nb127D01-HA or NbVHH05-HA. Scale bars: 20 µm.
    Figure Legend Snippet: Endogenous VHH05- or 127D01-tagging using CRISPR/Cas9. Workflow ( A ), fly embryos transformation ( B ), genotyping ( C ), and applications ( D ). ( A’ ) Common scarless vectors used for constructing donors. N- terminal vectors (PHD-N-3x127D01-donor or PHD-N-3xVHH05-donor) and C-terminal vectors (PHD-C-3x127D01-donor or PHD-C-3xVHH05-donor) contain EcoRI restriction enzyme sites that introduce the homologous arm sequences into donors. ( B’ ) Workflow example for introducing knock-in (KI) tags into the third chromosome. ( C’ ) Genotyping example of KI 3xVHH05 and 3x127D01 into the C-terminus of H2Av. Gel results showing the 5’ and 3’ PCR junctions. Representative sequencing chromatogram of PCR products from the junction PCR. ( D’ ) Immunostaining of H2Av- 3x127D01 and H2Av-3xVHH05. Adult male or female guts were dissected and stained with Nb127D01-HA or NbVHH05-HA. Scale bars: 20 µm.

    Techniques Used: CRISPR, Transformation Assay, Genotyping Assay, Introduce, Knock-In, Polymerase Chain Reaction, Sequencing, Immunostaining, Staining

    3) Product Images from "High-throughput screening of soluble recombinant proteins"

    Article Title: High-throughput screening of soluble recombinant proteins

    Journal: Protein Science : A Publication of the Protein Society

    doi:

    Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.
    Figure Legend Snippet: Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Ligation

    4) 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

    5) Product Images from "Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli"

    Article Title: Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119221

    Homologous recombination between vector and insert generated by restriction endonucleases. (A) The pNatMX was cleaved with the PvuII endonuclease generating the natMX fragment of 1469 bp. The pUC19 was prepared by digestion with the EcoRI and HindIII restriction enzymes, resulting in a 2639 bp linear plasmid. Homologous recombination between the natMX and pUC19 fragments generated the pUC19Nat plasmid. (B) Agarose gel electrophoresis after gel purification of the fragments natMX and pUC19. (C) The counting of colonies after transformation of the vector alone and co-transformation of the pUC19 plus the fragment natMX. (D) Colony PCR screening confirmed 100% positive cloning events. Abbreviations are as described in Fig. 2 .
    Figure Legend Snippet: Homologous recombination between vector and insert generated by restriction endonucleases. (A) The pNatMX was cleaved with the PvuII endonuclease generating the natMX fragment of 1469 bp. The pUC19 was prepared by digestion with the EcoRI and HindIII restriction enzymes, resulting in a 2639 bp linear plasmid. Homologous recombination between the natMX and pUC19 fragments generated the pUC19Nat plasmid. (B) Agarose gel electrophoresis after gel purification of the fragments natMX and pUC19. (C) The counting of colonies after transformation of the vector alone and co-transformation of the pUC19 plus the fragment natMX. (D) Colony PCR screening confirmed 100% positive cloning events. Abbreviations are as described in Fig. 2 .

    Techniques Used: Homologous Recombination, Plasmid Preparation, Generated, Agarose Gel Electrophoresis, Gel Purification, Transformation Assay, Polymerase Chain Reaction, Clone Assay

    6) 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

    7) 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

    8) Product Images from "Design and Characterization of Bioengineered Cancer-Like Stem Cells"

    Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141172

    Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
    Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Techniques Used: Subcloning, Clone Assay, DNA Sequencing

    9) 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

    Characterization of mutants of OV-IA82Δ113 and OV-IA82Δ116 by Southern blotting . A . OV-IA82Δ113. Upper panel is a schematic of OV-IA82 genome before and after removal of the 113 gene using the deletion vector, pSPV-EGFP by double homologous recombination to generate gene-deletion mutant OV-IA82Δ113. Lower panel shows Southern blot analysis. Genomic DNA was isolated from OV-IA82 (lane 1) and OV-IA82Δ113 (lane 2) and digested with restriction enzyme AflII or EcoRI respectively. The 113 internal probe was unable to detect the 113 gene in the recombinant OV-IA82Δ113 genome, indicating that the 113 gene was completely removed from the 113 locus of the genome. The 001 probe detected both end of the 001 loci in both OV-IA82 and OV-IA82Δ113 genomes. B . OV-IA82Δ116. Upper panel A is a schematic of OV-IA82 genome before and after removal of the 116 gene using the deletion vector, pSPV-EGFP by double homologous recombination to generate gene-deletion mutant OV-IA82Δ116. Lower panel shows the 116 gene, which was completely removed from the 116 locus of the OV-IA82 genome by Southern blot analysis. The 116 internal probe was unable to detect the 116 gene in the OV-IA82Δ116 genome. The 001 probe detected both end of sequences in OV-IA82 (lane: 1) and three different clones of OV-IA82Δ116 (lanes: 2 to 4).
    Figure Legend Snippet: Characterization of mutants of OV-IA82Δ113 and OV-IA82Δ116 by Southern blotting . A . OV-IA82Δ113. Upper panel is a schematic of OV-IA82 genome before and after removal of the 113 gene using the deletion vector, pSPV-EGFP by double homologous recombination to generate gene-deletion mutant OV-IA82Δ113. Lower panel shows Southern blot analysis. Genomic DNA was isolated from OV-IA82 (lane 1) and OV-IA82Δ113 (lane 2) and digested with restriction enzyme AflII or EcoRI respectively. The 113 internal probe was unable to detect the 113 gene in the recombinant OV-IA82Δ113 genome, indicating that the 113 gene was completely removed from the 113 locus of the genome. The 001 probe detected both end of the 001 loci in both OV-IA82 and OV-IA82Δ113 genomes. B . OV-IA82Δ116. Upper panel A is a schematic of OV-IA82 genome before and after removal of the 116 gene using the deletion vector, pSPV-EGFP by double homologous recombination to generate gene-deletion mutant OV-IA82Δ116. Lower panel shows the 116 gene, which was completely removed from the 116 locus of the OV-IA82 genome by Southern blot analysis. The 116 internal probe was unable to detect the 116 gene in the OV-IA82Δ116 genome. The 001 probe detected both end of sequences in OV-IA82 (lane: 1) and three different clones of OV-IA82Δ116 (lanes: 2 to 4).

    Techniques Used: Southern Blot, Plasmid Preparation, Homologous Recombination, Mutagenesis, Isolation, Recombinant, Clone Assay

    10) Product Images from "Bromodomain Protein Brd4 Plays a Key Role in Merkel Cell Polyomavirus DNA Replication"

    Article Title: Bromodomain Protein Brd4 Plays a Key Role in Merkel Cell Polyomavirus DNA Replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003021

    Brd4 is important for MCV DNA replication. A . Brd4 knockdown inhibits MCV replication in vivo . C33A cells were transfected with either a siRNA targeting Brd4 (K.D.) or a non-targeting siRNA control (C.O.). Forty-eight h later, cells were transfected with pT+Ori and this time was set as 0 h. Total DNA was extracted at 6, 24 and 42 h p.t.; 2 µ g of the DNA samples from 6 h p.t. were digested with EcoRI and plasmid DNA was detected by Southern blotting. 10 µ g of the DNA samples from 24 and 42 h p.t. were digested with both EcoRI and DpnI to specifically detect replicated plasmid. Protein extracts were immunoblotted for MCV LT, Brd4 and actin. B . MCV LT-dependent in vitro replication of MCV genome. In vitro MCV replication was performed using full length MCV genomic DNA and cell extracts prepared from 293T cells transfected with either pcDNA4C-MCV LT or pcDNA4C. C . Brd4 knockdown inhibits viral DNA synthesis in vitro and the inhibition can be rescued by recombinant Brd4. 293T cells were transfected with a Brd4 siRNA or a control siRNA. At 40 h p.t., cells were re-transfected with pcDNA4C-MCV LT. Cell extracts were prepared at 88 h p.t. and used for in vitro replication of MCV DNA. In the “rBrd4” condition, 3 µg His-Brd4 purified from insect cells using nickel resin was added to the Brd4 knockdown extract prior to performing the replication assay. In the “Control” condition, an equal amount of nonspecific proteins eluted from the nickel resin incubated with insect cells carrying wild-type baculovirus were used. All reactions were performed in triplicates. Immunoblots of cell extracts used in the assay and His-Brd4 purified from insect cells are shown in Fig. S3B and S3C . D . Brd4 knockdown inhibits MCV DNA replication in vitro and the inhibition can be rescued by Brd4 purified from mammalian cells. Extracts from cells transfected with a Brd4 siRNA and pcDNA4C-MCV LT as described in C were used in the in vitro replication assay. In the “Brd4” condition, 400 ng Brd4 purified from 293T cells was added to the Brd4 knockdown extract prior to performing the replication assay. In the “Control” condition, an equal amount of nonspecific proteins isolated from the vector control cells were used. All reactions were performed in triplicates. Coomassie Brilliant Blue staining of Brd4 purified from 293T cells are shown in Fig. S3E . E . Dose-dependent rescue of in vitro viral replication by the purified Brd4 protein. Extracts from cells transfected with a Brd4 siRNA and pcDNA4C-MCV LT as described in C were used in the in vitro replication assay. Increasing amounts of purified Brd4 was added to the reactions. All reactions were performed in triplicates.
    Figure Legend Snippet: Brd4 is important for MCV DNA replication. A . Brd4 knockdown inhibits MCV replication in vivo . C33A cells were transfected with either a siRNA targeting Brd4 (K.D.) or a non-targeting siRNA control (C.O.). Forty-eight h later, cells were transfected with pT+Ori and this time was set as 0 h. Total DNA was extracted at 6, 24 and 42 h p.t.; 2 µ g of the DNA samples from 6 h p.t. were digested with EcoRI and plasmid DNA was detected by Southern blotting. 10 µ g of the DNA samples from 24 and 42 h p.t. were digested with both EcoRI and DpnI to specifically detect replicated plasmid. Protein extracts were immunoblotted for MCV LT, Brd4 and actin. B . MCV LT-dependent in vitro replication of MCV genome. In vitro MCV replication was performed using full length MCV genomic DNA and cell extracts prepared from 293T cells transfected with either pcDNA4C-MCV LT or pcDNA4C. C . Brd4 knockdown inhibits viral DNA synthesis in vitro and the inhibition can be rescued by recombinant Brd4. 293T cells were transfected with a Brd4 siRNA or a control siRNA. At 40 h p.t., cells were re-transfected with pcDNA4C-MCV LT. Cell extracts were prepared at 88 h p.t. and used for in vitro replication of MCV DNA. In the “rBrd4” condition, 3 µg His-Brd4 purified from insect cells using nickel resin was added to the Brd4 knockdown extract prior to performing the replication assay. In the “Control” condition, an equal amount of nonspecific proteins eluted from the nickel resin incubated with insect cells carrying wild-type baculovirus were used. All reactions were performed in triplicates. Immunoblots of cell extracts used in the assay and His-Brd4 purified from insect cells are shown in Fig. S3B and S3C . D . Brd4 knockdown inhibits MCV DNA replication in vitro and the inhibition can be rescued by Brd4 purified from mammalian cells. Extracts from cells transfected with a Brd4 siRNA and pcDNA4C-MCV LT as described in C were used in the in vitro replication assay. In the “Brd4” condition, 400 ng Brd4 purified from 293T cells was added to the Brd4 knockdown extract prior to performing the replication assay. In the “Control” condition, an equal amount of nonspecific proteins isolated from the vector control cells were used. All reactions were performed in triplicates. Coomassie Brilliant Blue staining of Brd4 purified from 293T cells are shown in Fig. S3E . E . Dose-dependent rescue of in vitro viral replication by the purified Brd4 protein. Extracts from cells transfected with a Brd4 siRNA and pcDNA4C-MCV LT as described in C were used in the in vitro replication assay. Increasing amounts of purified Brd4 was added to the reactions. All reactions were performed in triplicates.

    Techniques Used: In Vivo, Transfection, Plasmid Preparation, Southern Blot, In Vitro, DNA Synthesis, Inhibition, Recombinant, Purification, Incubation, Western Blot, Isolation, Staining

    The DNI inhibits MCV DNA replication. A . DNI expression reduces MCV genome replication in an MCV sT/LT stable cell line. 293-4T cells were transfected with the MCV genome. At 84 h p.t., cells were re-transfected with either pcDNA4C-Brd4 471-730 (DNI) or pcDNA4C (control). The time for the second transfection was set as 0 hr. Viral genomes present in cellular DNA extracted at different time points were quantified using qPCR. Viral genome copies were normalized to beta-actin DNA and presented as a percentage of the viral genome detected at 0 hr. Mean and standard deviation were calculated from three independent experiments. B . FACS cell cycle analysis. 293T cells were transfected with pcDNA4C (Vec) or pcDNA4C-Brd4 471-730 (DNI) either alone or together with re-ligated MCV genome (MCV) as indicated. At 48 h p.t., cells were fixed and subjected to FACS analysis. C . The DNI inhibits autonomous MCV replication. 293T cells were co-transfected with 1 µg MCV genome and either 0, 7, or 9 µg of pcDNA4C-Brd4 471-730 (DNI). 2 µg of cellular DNA from 6 h p.t. was digested with EcoRI to show that equal amount of viral genomes were transfected into cells. 10 µg of DNA extracted from 24 and 42 h p.t. was digested with EcoRI and DpnI to detect the replicated viral DNA. The viral DNA was analyzed using Southern blotting. Intensities of autoradiography signal were analyzed using ImageJ and normalized to the value obtained with 0 µg of DNI at each time point. Protein extracts were immunoblotted for Xpress-DNI, MCV LT and actin. A schematic diagram of MCV replication time-course is also shown. D . The DNI inhibits viral DNA synthesis in vitro . 293T cells were co-transfected with pcDNA4C-MCV LT and pcDNA4C-Brd4. Cellular extracts were supplemented with increasing amount of recombinant DNI and used in the in vitro MCV replication assay. All reactions were performed in triplicates. DNA from the same reactions omitting [α- 32 P] dCTP and creatine kinase were resolved on an agarose gel and stained with ethidium bromide. E . The DNI does not inhibit SV40 replication in vitro . In vitro replication using 293T cell extracts and pEGFP-C1 (carrying SV40 Ori) as template was performed as described in D . Increasing amount of recombinant DNI was added to the in vitro replication assays.
    Figure Legend Snippet: The DNI inhibits MCV DNA replication. A . DNI expression reduces MCV genome replication in an MCV sT/LT stable cell line. 293-4T cells were transfected with the MCV genome. At 84 h p.t., cells were re-transfected with either pcDNA4C-Brd4 471-730 (DNI) or pcDNA4C (control). The time for the second transfection was set as 0 hr. Viral genomes present in cellular DNA extracted at different time points were quantified using qPCR. Viral genome copies were normalized to beta-actin DNA and presented as a percentage of the viral genome detected at 0 hr. Mean and standard deviation were calculated from three independent experiments. B . FACS cell cycle analysis. 293T cells were transfected with pcDNA4C (Vec) or pcDNA4C-Brd4 471-730 (DNI) either alone or together with re-ligated MCV genome (MCV) as indicated. At 48 h p.t., cells were fixed and subjected to FACS analysis. C . The DNI inhibits autonomous MCV replication. 293T cells were co-transfected with 1 µg MCV genome and either 0, 7, or 9 µg of pcDNA4C-Brd4 471-730 (DNI). 2 µg of cellular DNA from 6 h p.t. was digested with EcoRI to show that equal amount of viral genomes were transfected into cells. 10 µg of DNA extracted from 24 and 42 h p.t. was digested with EcoRI and DpnI to detect the replicated viral DNA. The viral DNA was analyzed using Southern blotting. Intensities of autoradiography signal were analyzed using ImageJ and normalized to the value obtained with 0 µg of DNI at each time point. Protein extracts were immunoblotted for Xpress-DNI, MCV LT and actin. A schematic diagram of MCV replication time-course is also shown. D . The DNI inhibits viral DNA synthesis in vitro . 293T cells were co-transfected with pcDNA4C-MCV LT and pcDNA4C-Brd4. Cellular extracts were supplemented with increasing amount of recombinant DNI and used in the in vitro MCV replication assay. All reactions were performed in triplicates. DNA from the same reactions omitting [α- 32 P] dCTP and creatine kinase were resolved on an agarose gel and stained with ethidium bromide. E . The DNI does not inhibit SV40 replication in vitro . In vitro replication using 293T cell extracts and pEGFP-C1 (carrying SV40 Ori) as template was performed as described in D . Increasing amount of recombinant DNI was added to the in vitro replication assays.

    Techniques Used: Expressing, Stable Transfection, Transfection, Real-time Polymerase Chain Reaction, Standard Deviation, FACS, Cell Cycle Assay, Southern Blot, Autoradiography, DNA Synthesis, In Vitro, Recombinant, Agarose Gel Electrophoresis, Staining

    11) Product Images from "Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint"

    Article Title: Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1283

    Analysis of the positions of left-handed DNA in the LR-chimeras (8% PAGE). ( A ) Cleavage (5 min) of lc-74 0 and cc-74 0 . Lane 2: lc-74 0 ; lanes 3 and 4: lc-74 0 cleaved by SphI and MobI, respectively; lane 5: cc-74 0 ; lanes 6,7: cc-74 0 cleaved by SphI and MobI, respectively. ( B ) Schematic illustration of lc-74 0 and linear products of cleavage by SphI and MobI, respectively. ( C ) Cleavage (5 min) of lc-74 1 and cc-74 1 . Lane 2: lc-74 1 ; lanes 3–5: lc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively; lane 6: cc-74 1 ; lanes 7–9: cc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively. ( D ) Schematic illustration of lc-74 1 , lc-74 1EcoRI and linear products of cleavage by EcoRI, MobI, and Hpych4Iv, respectively. ( E ) Cleavage (30 min) of cc-74 0 and cc-74 1 after binding to Z22. Lane 1: Z22 bind to cc-74 0 ; lanes 2, 3: Cleavage of cc-74 0 by SphI and MboI after binding to Z22, respectively; lane 4: Z22 bind to cc-74 1 ; lanes 5–7: Cleavage of cc-74 1 by EcoRI, MboI, and Hpych4Iv after binding to Z22, respectively.
    Figure Legend Snippet: Analysis of the positions of left-handed DNA in the LR-chimeras (8% PAGE). ( A ) Cleavage (5 min) of lc-74 0 and cc-74 0 . Lane 2: lc-74 0 ; lanes 3 and 4: lc-74 0 cleaved by SphI and MobI, respectively; lane 5: cc-74 0 ; lanes 6,7: cc-74 0 cleaved by SphI and MobI, respectively. ( B ) Schematic illustration of lc-74 0 and linear products of cleavage by SphI and MobI, respectively. ( C ) Cleavage (5 min) of lc-74 1 and cc-74 1 . Lane 2: lc-74 1 ; lanes 3–5: lc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively; lane 6: cc-74 1 ; lanes 7–9: cc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively. ( D ) Schematic illustration of lc-74 1 , lc-74 1EcoRI and linear products of cleavage by EcoRI, MobI, and Hpych4Iv, respectively. ( E ) Cleavage (30 min) of cc-74 0 and cc-74 1 after binding to Z22. Lane 1: Z22 bind to cc-74 0 ; lanes 2, 3: Cleavage of cc-74 0 by SphI and MboI after binding to Z22, respectively; lane 4: Z22 bind to cc-74 1 ; lanes 5–7: Cleavage of cc-74 1 by EcoRI, MboI, and Hpych4Iv after binding to Z22, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Binding Assay

    12) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    13) 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

    14) 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

    15) Product Images from "Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome"

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190177

    Characterization of RiCF. (A) Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone. (B) Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM. (C) RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE. (D) Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented. (E) A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis. (F) Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.
    Figure Legend Snippet: Characterization of RiCF. (A) Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone. (B) Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM. (C) RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE. (D) Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented. (E) A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis. (F) Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.

    Techniques Used: Lysis, Incubation, Electrophoresis

    16) 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

    17) Product Images from "Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13"

    Article Title: Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006232

    Southern blot analyses of C. perfringens random mutants obtained after electroporation with EZ-Tn 5 transposomes. After selection on BHI plates containing Erm (40 µg/ml), DNA was extracted from strain 13 transformants. Following digestion with EcoRI (A) or XbaI (B), the digested DNA was electrophoresed and blotted to a nylon membrane. DNA on the membranes was then hybridized with a Dig-labeled erm probe, as found in the C. perfringens -modified EZ-Tn5, and blots were developed as described in the Materials and Methods . Size of DNA fragments, in kilobases (kb), is shown at left.
    Figure Legend Snippet: Southern blot analyses of C. perfringens random mutants obtained after electroporation with EZ-Tn 5 transposomes. After selection on BHI plates containing Erm (40 µg/ml), DNA was extracted from strain 13 transformants. Following digestion with EcoRI (A) or XbaI (B), the digested DNA was electrophoresed and blotted to a nylon membrane. DNA on the membranes was then hybridized with a Dig-labeled erm probe, as found in the C. perfringens -modified EZ-Tn5, and blots were developed as described in the Materials and Methods . Size of DNA fragments, in kilobases (kb), is shown at left.

    Techniques Used: Southern Blot, Electroporation, Selection, Labeling, Modification

    Generation of a C. perfringens agrB mutant and complementing strains. A) Southern blot analyses, as described in Fig. 2 , using EcoRI-digested DNA from CPJV501 and a Dig-labeled probe that detected a single copy of the erm gene. Size of DNA fragments, in kilobases (kb) is shown at left. B) PCR was performed with DNA extracted from the indicated strain and the following pair of primers, agrBFwd and agrBRev in reactions containing DNA from strain 13 (S13), CPJV501 and CPJVp1; agrBFwd and argDR for CPJVp2 and agrF1 and agrD100R for CPJVp3. DNA ladders (100 bp or 1 kb) were included in the first and last lane of the gel. Asterisks show the expected PCR product when the primers amplified the Tn5-disprupted agr B gene. C) Genes cloned in the E. coli-C. perfringens shuttle plasmid pJIR750 to complement the agr B transposon mutant. As shown, P1 encodes the agr B gene alone, P2 the agr B and agr D genes and P3 encodes two-genes (CPE1562 and CPE1563) upstream the agr B gene (CPE1561) and agr B and agr D.
    Figure Legend Snippet: Generation of a C. perfringens agrB mutant and complementing strains. A) Southern blot analyses, as described in Fig. 2 , using EcoRI-digested DNA from CPJV501 and a Dig-labeled probe that detected a single copy of the erm gene. Size of DNA fragments, in kilobases (kb) is shown at left. B) PCR was performed with DNA extracted from the indicated strain and the following pair of primers, agrBFwd and agrBRev in reactions containing DNA from strain 13 (S13), CPJV501 and CPJVp1; agrBFwd and argDR for CPJVp2 and agrF1 and agrD100R for CPJVp3. DNA ladders (100 bp or 1 kb) were included in the first and last lane of the gel. Asterisks show the expected PCR product when the primers amplified the Tn5-disprupted agr B gene. C) Genes cloned in the E. coli-C. perfringens shuttle plasmid pJIR750 to complement the agr B transposon mutant. As shown, P1 encodes the agr B gene alone, P2 the agr B and agr D genes and P3 encodes two-genes (CPE1562 and CPE1563) upstream the agr B gene (CPE1561) and agr B and agr D.

    Techniques Used: Mutagenesis, Southern Blot, Labeling, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation

    18) 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

    19) Product Images from "Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy"

    Article Title: Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0260837

    Southern blot of peripheral blood leukocyte and primary HCEC lines established from control and FECD patients. (A) TCF4 repeats in DNA isolated from peripheral blood leukocytes (Lanes 1–3) and primary HCECs (Lanes 4–6). Lanes 1 [Cont(4)] and 4 [Cont(5)] represent samples obtained from different control patients. Lanes 2, 5 [FECD(2)] and 3, 6 [FECD(1)] are samples from two FECD patients with expanded repeats. Arrows indicate large molecular weight bands in FECD1 and FECD2 corresponding to approximately 15,000 base pairs and approximately 4500 repeats. For analysis, 5 μg leukocyte DNA and 20 μg primary HCEC DNA was digested with EcoRI and separated on an 0.8% agarose gel. (B) Southern blot of control and FECD primary HCEC cell lines showing large molecular weight bands ranging from approximately 5500— > 15,000 base pairs. This corresponds to all FECD samples having at least one allele with a TCF4 expansion of > 1100 repeats. Genotype of TCF4 expansion size as determined in leukocytes for each FECD HCEC line is as follows: FECD(4)—16/69; FECD(5)—25/71; FECD(6)—73/82; FECD(7)—18/52; FECD(8)—12/64; FECD(9)—12/66; FECD(10)—100/123; and FECD(11)—17/98. Note that FECD(2) was utilized as a positive control in both (A) and (B). For analysis, 5 μg of primary HCEC DNA was digested with EcoRI and separated on an 0.8% agarose gel.
    Figure Legend Snippet: Southern blot of peripheral blood leukocyte and primary HCEC lines established from control and FECD patients. (A) TCF4 repeats in DNA isolated from peripheral blood leukocytes (Lanes 1–3) and primary HCECs (Lanes 4–6). Lanes 1 [Cont(4)] and 4 [Cont(5)] represent samples obtained from different control patients. Lanes 2, 5 [FECD(2)] and 3, 6 [FECD(1)] are samples from two FECD patients with expanded repeats. Arrows indicate large molecular weight bands in FECD1 and FECD2 corresponding to approximately 15,000 base pairs and approximately 4500 repeats. For analysis, 5 μg leukocyte DNA and 20 μg primary HCEC DNA was digested with EcoRI and separated on an 0.8% agarose gel. (B) Southern blot of control and FECD primary HCEC cell lines showing large molecular weight bands ranging from approximately 5500— > 15,000 base pairs. This corresponds to all FECD samples having at least one allele with a TCF4 expansion of > 1100 repeats. Genotype of TCF4 expansion size as determined in leukocytes for each FECD HCEC line is as follows: FECD(4)—16/69; FECD(5)—25/71; FECD(6)—73/82; FECD(7)—18/52; FECD(8)—12/64; FECD(9)—12/66; FECD(10)—100/123; and FECD(11)—17/98. Note that FECD(2) was utilized as a positive control in both (A) and (B). For analysis, 5 μg of primary HCEC DNA was digested with EcoRI and separated on an 0.8% agarose gel.

    Techniques Used: Southern Blot, Isolation, Molecular Weight, Agarose Gel Electrophoresis, Positive Control

    20) 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

    21) 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

    22) Product Images from "Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster"

    Article Title: Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster

    Journal: bioRxiv

    doi: 10.1101/2021.04.15.440022

    ME, EB, and ey -PRE interact in long-range to regulate ey locus. (a) Interaction of ME with various regions at ey locus. 3C was performed in 0-16 h embryo using EcoRI restriction digestion followed by ligation. Ligated hybrids were PCR amplified (cross linked) using all forward primer pairs and confirmed by sequencing. A control template was generated by EcoRI restriction digestion and ligation of equimolar mix of PCR products of all regions flanking to the restriction sites. Primer efficiency of all primers were observed by PCR using the control template (control). Interaction frequencies mean and standard deviations (b-vii, ME- EcoRI) were calculated by gel quantification of three replicates, as described ( N aumova et al . 2012 ). EcoRI fragment that contains ME (eyE1) interacts with ey - PRE (eyE3) and with an internal region (eyE4) and EB (eyE6) of ey (red lines in b-v). However, such interactions are not observed with upstream regions towards myo (myoE-3, myoE-2 and myoE-1), other internal regions of ey (eyE2 and eyE5) and in farther downstream regions of bt (btE1 and btE2) (grey lines in b-v). (b) Comparative analysis of chromatin interaction at ey locus. (i ii) A merge Hi-C heatmap of chromatin interactions in ∼250 Kb region of the fourth chromosome along with TAD classification, chromatin states and genes in Kc167 cells. A merged Hi-C data from Li et al. Cubenas et al. ( L i et al . 2015 ; C ubenas-potts et al . 2017 ) was visualized using pyGenomeTracks ( L opez-delisle et al . 2021 ) and chromatin loops were inferred from the same (shaded triangles). The TAD classification track contains the four classifications from ref ( R amirez et al . 2018 ) : Active TAD, yellow; Inactive TAD, black; Inactive TAD contains blue-PcGand green-HP1. The chromatin state track shows five chromatin types from ref ( F ilion et al . 2010 ) that includes: active chromatin-red and yellow; inactive chromatin-black; PcG-blue; HP1-green. The two genes ey and bt contain PcG mediated inactive chromatin feature that demarcates a PcG TAD. However, ey and bt genes appear to be in separate sub-domain within the TAD. (iii) Genes and regulatory region of ey . (iv) EcoRI/DpnII sites and uni- directional primers used for 3C. (v) Regions interacting to ME inferred from 3C are marked with red looping lines and non-interacting regions are marked with grey looping lines. (vi) Relative interaction frequencies of EcoRI fragment containing ME to other EcoRI fragments (ME-EcoRI). Relative interaction frequencies of DpnII fragment containing ME (ME-DpnII) and EB (EB-DpnII) with other DpnII fragments . At all the data point’s, mean and standard deviation were generated from 3 experiments. The significant differences to adjacent fragments were assessed by one-way ANOVA using Fisher LSD test, α=0.05. Together 3C and Hi-C data suggest ME and EB interacts in long range to demarcate the ey domain.
    Figure Legend Snippet: ME, EB, and ey -PRE interact in long-range to regulate ey locus. (a) Interaction of ME with various regions at ey locus. 3C was performed in 0-16 h embryo using EcoRI restriction digestion followed by ligation. Ligated hybrids were PCR amplified (cross linked) using all forward primer pairs and confirmed by sequencing. A control template was generated by EcoRI restriction digestion and ligation of equimolar mix of PCR products of all regions flanking to the restriction sites. Primer efficiency of all primers were observed by PCR using the control template (control). Interaction frequencies mean and standard deviations (b-vii, ME- EcoRI) were calculated by gel quantification of three replicates, as described ( N aumova et al . 2012 ). EcoRI fragment that contains ME (eyE1) interacts with ey - PRE (eyE3) and with an internal region (eyE4) and EB (eyE6) of ey (red lines in b-v). However, such interactions are not observed with upstream regions towards myo (myoE-3, myoE-2 and myoE-1), other internal regions of ey (eyE2 and eyE5) and in farther downstream regions of bt (btE1 and btE2) (grey lines in b-v). (b) Comparative analysis of chromatin interaction at ey locus. (i ii) A merge Hi-C heatmap of chromatin interactions in ∼250 Kb region of the fourth chromosome along with TAD classification, chromatin states and genes in Kc167 cells. A merged Hi-C data from Li et al. Cubenas et al. ( L i et al . 2015 ; C ubenas-potts et al . 2017 ) was visualized using pyGenomeTracks ( L opez-delisle et al . 2021 ) and chromatin loops were inferred from the same (shaded triangles). The TAD classification track contains the four classifications from ref ( R amirez et al . 2018 ) : Active TAD, yellow; Inactive TAD, black; Inactive TAD contains blue-PcGand green-HP1. The chromatin state track shows five chromatin types from ref ( F ilion et al . 2010 ) that includes: active chromatin-red and yellow; inactive chromatin-black; PcG-blue; HP1-green. The two genes ey and bt contain PcG mediated inactive chromatin feature that demarcates a PcG TAD. However, ey and bt genes appear to be in separate sub-domain within the TAD. (iii) Genes and regulatory region of ey . (iv) EcoRI/DpnII sites and uni- directional primers used for 3C. (v) Regions interacting to ME inferred from 3C are marked with red looping lines and non-interacting regions are marked with grey looping lines. (vi) Relative interaction frequencies of EcoRI fragment containing ME to other EcoRI fragments (ME-EcoRI). Relative interaction frequencies of DpnII fragment containing ME (ME-DpnII) and EB (EB-DpnII) with other DpnII fragments . At all the data point’s, mean and standard deviation were generated from 3 experiments. The significant differences to adjacent fragments were assessed by one-way ANOVA using Fisher LSD test, α=0.05. Together 3C and Hi-C data suggest ME and EB interacts in long range to demarcate the ey domain.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Hi-C, Standard Deviation

    23) Product Images from "Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity"

    Article Title: Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.03.028

    Characterization of shAAV Genomes and In Vivo Evaluation of shAAV Vectors (A) Schematic of pCis constructs used for AAV production. The mTR was removed from vector constructs to assess the ability of shDNA sequences to create double-stranded shAAV vectors. (B) The predicted sizes of packaged genomes were calculated from the base pair lengths between shDNA sequences and wtTR. (C) Viral genome DNA from purified vectors (∼1.0 × 10 10 GCs) in native (left panel) and alkaline (right panel) agarose gels. (D) EGFP expression in livers of adult mice 3 weeks after intravenous injection of rAAV (1.6 × 10 13 GCs/kg). (E) Southern blot analysis of EcoRI- or MscI-digested liver DNA using an EGFP probe. The MscI site is denoted in (A). Small black arrows, linear rAAV genomes; purple arrows, circular rAAVs; magenta arrows, linearized circular rAAVs; white arrowheads, digested linear rAAVs.
    Figure Legend Snippet: Characterization of shAAV Genomes and In Vivo Evaluation of shAAV Vectors (A) Schematic of pCis constructs used for AAV production. The mTR was removed from vector constructs to assess the ability of shDNA sequences to create double-stranded shAAV vectors. (B) The predicted sizes of packaged genomes were calculated from the base pair lengths between shDNA sequences and wtTR. (C) Viral genome DNA from purified vectors (∼1.0 × 10 10 GCs) in native (left panel) and alkaline (right panel) agarose gels. (D) EGFP expression in livers of adult mice 3 weeks after intravenous injection of rAAV (1.6 × 10 13 GCs/kg). (E) Southern blot analysis of EcoRI- or MscI-digested liver DNA using an EGFP probe. The MscI site is denoted in (A). Small black arrows, linear rAAV genomes; purple arrows, circular rAAVs; magenta arrows, linearized circular rAAVs; white arrowheads, digested linear rAAVs.

    Techniques Used: In Vivo, Construct, Plasmid Preparation, Purification, Expressing, Mouse Assay, Injection, Southern Blot

    24) 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

    25) Product Images from "Structural Basis for Shelterin Bridge Assembly"

    Article Title: Structural Basis for Shelterin Bridge Assembly

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.10.032

    The “conformational trigger” in Poz1 is essential for shelterin assembly and telomere length regulation ( A ) Telomere length analysis of poz1-NTD mutant cells from successive re-streaks on agar plates. Total genomic DNA was digested by EcoRI. Wild-type cells are denoted as “WT” in the blot. Simultaneously digested pol1 + DNA fragment serves as the loading control. Telomeres are elongated in poz1-ΔN and poz1-L14R cells (colored in red). ( B ) Bio-Layer Interferometry (BLI) sensorgrams monitoring dissociation and association events in real time between Poz1 L14R -Tpz1 475–508 and Rap1 446-512 using Octet red96 (R 2 =0.9949). BLI experiments were repeated twice and representative results were shown. ( C ) and ( D ) Co-IP assays evaluating the effect of Poz1 N-terminal helix deletion or mutation on Taz1-Rap1, Poz1-Rap1 (C), and Tpz1-Poz1 (D) interactions. Poz1-Rap1 interaction is fully disrupted in poz1-ΔN and poz1-L14R cells, whereas Poz1-Tpz1 interaction is weakened in poz1-ΔN and poz1-L14R cells. Rap1-Taz1 interaction remains unchanged. ( E ), (F) and (G) Telomeric localization of Rap1 (E), Poz1 (F), and Tpz1 (G) in strains with Poz1 N-terminal helix deletion or mutation was monitored by chromatin immunoprecipitation (ChIP) assay. Slot blot hybridized with telomere probe was used to visualize the telomeric signal associated with each protein. Error bars in the quantitation of the slot-blot analysis represent standard deviations of three individual repeats. Each ChIP assay was performed in triplicate (n=3). Error bars represent standard deviations. .
    Figure Legend Snippet: The “conformational trigger” in Poz1 is essential for shelterin assembly and telomere length regulation ( A ) Telomere length analysis of poz1-NTD mutant cells from successive re-streaks on agar plates. Total genomic DNA was digested by EcoRI. Wild-type cells are denoted as “WT” in the blot. Simultaneously digested pol1 + DNA fragment serves as the loading control. Telomeres are elongated in poz1-ΔN and poz1-L14R cells (colored in red). ( B ) Bio-Layer Interferometry (BLI) sensorgrams monitoring dissociation and association events in real time between Poz1 L14R -Tpz1 475–508 and Rap1 446-512 using Octet red96 (R 2 =0.9949). BLI experiments were repeated twice and representative results were shown. ( C ) and ( D ) Co-IP assays evaluating the effect of Poz1 N-terminal helix deletion or mutation on Taz1-Rap1, Poz1-Rap1 (C), and Tpz1-Poz1 (D) interactions. Poz1-Rap1 interaction is fully disrupted in poz1-ΔN and poz1-L14R cells, whereas Poz1-Tpz1 interaction is weakened in poz1-ΔN and poz1-L14R cells. Rap1-Taz1 interaction remains unchanged. ( E ), (F) and (G) Telomeric localization of Rap1 (E), Poz1 (F), and Tpz1 (G) in strains with Poz1 N-terminal helix deletion or mutation was monitored by chromatin immunoprecipitation (ChIP) assay. Slot blot hybridized with telomere probe was used to visualize the telomeric signal associated with each protein. Error bars in the quantitation of the slot-blot analysis represent standard deviations of three individual repeats. Each ChIP assay was performed in triplicate (n=3). Error bars represent standard deviations. .

    Techniques Used: Mutagenesis, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Dot Blot, Quantitation Assay

    26) Product Images from "Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics"

    Article Title: Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096793

    Schematic overview of the library preparation process. Both RAD-tag (left) and whole-genome shotgun (right) library preparation methods start the same way, and diverge only at the final stage. (a) DNA is heated to denature the template strands. (b) Terminal deoxynucleotidyl transferase (TdT) is used to add a riboguanidine tail of a determined length [44] . (c) Priming with the Illumina P2 adaptor sequence, the Klenow exo- fragment generates the second strand. At this point, T4 DNA polymerase treatment is necessary to blunt the DNA fragments. After (d’) for RAD-tag sequencing, EcoRI is used to digest a subset of the fragments. (d’’ and e) a final ligation step adds the P1 Illumina adaptor sequence. Barcodes are ligated in-line, upstream of the read one sequencing primer binding site. After ligation of the final adaptor sequence, fragments are PCR-amplified to complete the sequencing adaptor. All libraries contained in-line barcodes in front of the read one sequencing site.
    Figure Legend Snippet: Schematic overview of the library preparation process. Both RAD-tag (left) and whole-genome shotgun (right) library preparation methods start the same way, and diverge only at the final stage. (a) DNA is heated to denature the template strands. (b) Terminal deoxynucleotidyl transferase (TdT) is used to add a riboguanidine tail of a determined length [44] . (c) Priming with the Illumina P2 adaptor sequence, the Klenow exo- fragment generates the second strand. At this point, T4 DNA polymerase treatment is necessary to blunt the DNA fragments. After (d’) for RAD-tag sequencing, EcoRI is used to digest a subset of the fragments. (d’’ and e) a final ligation step adds the P1 Illumina adaptor sequence. Barcodes are ligated in-line, upstream of the read one sequencing primer binding site. After ligation of the final adaptor sequence, fragments are PCR-amplified to complete the sequencing adaptor. All libraries contained in-line barcodes in front of the read one sequencing site.

    Techniques Used: Sequencing, Ligation, Binding Assay, Polymerase Chain Reaction, Amplification

    27) Product Images from "Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates"

    Article Title: Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.2069-2074.2004

    Comparison of the genetic organizations of AR-2 and AR-11. Double-headed striped arrows indicate the position of the rmtA locus and that of the region common to both sequenced areas. Inserts of pBCRMTH2, pBCRMTE2, and pBCRMTE11 are indicated by horizontal lines. Rectangles filled with wavy lines, sequences similar to part of Tn 5041 . Solid arrowheads in the 15.8-kbp EcoRI fragment, terminal inverted repeats. mer , the mercury resistance operon, includes merR . Sequence 1, transposase gene-like sequence; sequence 2, Na + /H + antiporter-like sequence; orfA , probable tRNA ribosyltransferase gene; orfQ ′, part of orfQ ; orfA ′, part of orfA ; IR, probable inverted repeat. Restriction sites: H, HindIII; E, EcoRI. Sequences 1 and 2 encode no complete proteins due to several frameshifts and deletions.
    Figure Legend Snippet: Comparison of the genetic organizations of AR-2 and AR-11. Double-headed striped arrows indicate the position of the rmtA locus and that of the region common to both sequenced areas. Inserts of pBCRMTH2, pBCRMTE2, and pBCRMTE11 are indicated by horizontal lines. Rectangles filled with wavy lines, sequences similar to part of Tn 5041 . Solid arrowheads in the 15.8-kbp EcoRI fragment, terminal inverted repeats. mer , the mercury resistance operon, includes merR . Sequence 1, transposase gene-like sequence; sequence 2, Na + /H + antiporter-like sequence; orfA , probable tRNA ribosyltransferase gene; orfQ ′, part of orfQ ; orfA ′, part of orfA ; IR, probable inverted repeat. Restriction sites: H, HindIII; E, EcoRI. Sequences 1 and 2 encode no complete proteins due to several frameshifts and deletions.

    Techniques Used: Sequencing

    28) 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

    29) 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

    30) Product Images from "Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a"

    Article Title: Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

    Journal: bioRxiv

    doi: 10.1101/496703

    PCR amplification and digestion and ligation overview for plasmid synthesis used in this study. Linkers including restriction sites are colored red, blue, or green. A) PCR products for cloning were amplified by amplification at a lower initial temperature to allow the incorporation of additional restriction sites on the 5’ end of the primers (shown in red and blue). The annealing temperature was then increased to amplify full-length products with the added restriction sites. B) Addition of the F. alni ACN14a nif cluster promoter region to the egfp coding sequence. Both the promoter region and egfp coding sequence were amplified with EcoRI sites (blue). These were then digested and ligated together followed by amplification of the ligation product. Restriction sites on the ends (red and green) were digested, allowing incorporation of the nif promoter: egfp product into the digested plasmid.
    Figure Legend Snippet: PCR amplification and digestion and ligation overview for plasmid synthesis used in this study. Linkers including restriction sites are colored red, blue, or green. A) PCR products for cloning were amplified by amplification at a lower initial temperature to allow the incorporation of additional restriction sites on the 5’ end of the primers (shown in red and blue). The annealing temperature was then increased to amplify full-length products with the added restriction sites. B) Addition of the F. alni ACN14a nif cluster promoter region to the egfp coding sequence. Both the promoter region and egfp coding sequence were amplified with EcoRI sites (blue). These were then digested and ligated together followed by amplification of the ligation product. Restriction sites on the ends (red and green) were digested, allowing incorporation of the nif promoter: egfp product into the digested plasmid.

    Techniques Used: Polymerase Chain Reaction, Amplification, Ligation, Plasmid Preparation, Clone Assay, Sequencing

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    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 <t>NheI</t> and <t>EcoRI</t>
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    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

    Journal: Nature protocols

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

    doi: 10.1038/nprot.2015.094

    Figure Lengend 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

    Article Snippet: Cycle Pure kit (Omega Bio-Tek, cat. no. D6492-02) QIAprep spin miniprep kit (Qiagen, cat. no.27104) EcoRI (New England BioLabs, cat. no. R0101S) NheI (New England BioLabs, cat. no. R0131S) Shrimp alkaline phosphatase (New England BioLabs, cat. no. R0371S) Adenosine 5′-triphosphate (ATP; New England BioLabs, cat. no. R0756S) T4 polynucleotide kinase (T4 PNK; New England BioLabs, cat. no. R0201S) T4 DNA ligase (New England BioLabs, cat. no. R0202L) Nt.BstNBI (New England BioLabs, cat. no. R0607L) Ethidium bromide (Sigma-Aldrich, cat. no. E1510) !

    Techniques: Plasmid Preparation

    Endogenous VHH05- or 127D01-tagging using CRISPR/Cas9. Workflow ( A ), fly embryos transformation ( B ), genotyping ( C ), and applications ( D ). ( A’ ) Common scarless vectors used for constructing donors. N- terminal vectors (PHD-N-3x127D01-donor or PHD-N-3xVHH05-donor) and C-terminal vectors (PHD-C-3x127D01-donor or PHD-C-3xVHH05-donor) contain EcoRI restriction enzyme sites that introduce the homologous arm sequences into donors. ( B’ ) Workflow example for introducing knock-in (KI) tags into the third chromosome. ( C’ ) Genotyping example of KI 3xVHH05 and 3x127D01 into the C-terminus of H2Av. Gel results showing the 5’ and 3’ PCR junctions. Representative sequencing chromatogram of PCR products from the junction PCR. ( D’ ) Immunostaining of H2Av- 3x127D01 and H2Av-3xVHH05. Adult male or female guts were dissected and stained with Nb127D01-HA or NbVHH05-HA. Scale bars: 20 µm.

    Journal: bioRxiv

    Article Title: Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

    doi: 10.1101/2021.04.16.440240

    Figure Lengend Snippet: Endogenous VHH05- or 127D01-tagging using CRISPR/Cas9. Workflow ( A ), fly embryos transformation ( B ), genotyping ( C ), and applications ( D ). ( A’ ) Common scarless vectors used for constructing donors. N- terminal vectors (PHD-N-3x127D01-donor or PHD-N-3xVHH05-donor) and C-terminal vectors (PHD-C-3x127D01-donor or PHD-C-3xVHH05-donor) contain EcoRI restriction enzyme sites that introduce the homologous arm sequences into donors. ( B’ ) Workflow example for introducing knock-in (KI) tags into the third chromosome. ( C’ ) Genotyping example of KI 3xVHH05 and 3x127D01 into the C-terminus of H2Av. Gel results showing the 5’ and 3’ PCR junctions. Representative sequencing chromatogram of PCR products from the junction PCR. ( D’ ) Immunostaining of H2Av- 3x127D01 and H2Av-3xVHH05. Adult male or female guts were dissected and stained with Nb127D01-HA or NbVHH05-HA. Scale bars: 20 µm.

    Article Snippet: For pW10-NbVHH05-HA, pW10-Nb127D01-HA, pW10-NbVHH05- GFP, pW10-Nb127D01-GFP, pW10-BiP-NbVHH05-HA, pW10-BiP-Nb127D01-HA, pW10-BiP-NbVHH05-GFP, and pW10-BiP-Nb127D01-GFP, pW10 was first linearized with EcoRI (NEB, R0101) and XbaI (NEB, R0145).

    Techniques: CRISPR, Transformation Assay, Genotyping Assay, Introduce, Knock-In, Polymerase Chain Reaction, Sequencing, Immunostaining, Staining

    Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: High-throughput screening of soluble recombinant proteins

    doi:

    Figure Lengend Snippet: Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.

    Article Snippet: To prepare vectors for ligation reactions, the vectors were restriction digested with EcoRI and XhoI and then dephosphorylated with calf intestinal alkaline phosphotase (New England Biolabs).

    Techniques: Clone Assay, Polymerase Chain Reaction, Ligation

    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.

    Journal: mBio

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

    doi: 10.1128/mBio.02674-18

    Figure Lengend 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.

    Article Snippet: Briefly, purified genomic DNA from each strain was digested with EcoRI (New England Biolabs), electrophoresed on an agarose gel, and transferred to a positively charged nylon membrane (Roche) using alkali transfer.

    Techniques: 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.

    Journal: mBio

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

    doi: 10.1128/mBio.02674-18

    Figure Lengend 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.

    Article Snippet: Briefly, purified genomic DNA from each strain was digested with EcoRI (New England Biolabs), electrophoresed on an agarose gel, and transferred to a positively charged nylon membrane (Roche) using alkali transfer.

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