ecori  (New England Biolabs)


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    EcoRI
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    EcoRI 50 000 units
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    R0101L
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    Category:
    Restriction Enzymes
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    50 000 units
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    New England Biolabs ecori
    EcoRI
    EcoRI 50 000 units
    https://www.bioz.com/result/ecori/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ecori - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    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 "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

    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 "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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    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 "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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    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 "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

    26) 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

    27) 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

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

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

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.062082606

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr078

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

    Techniques Used: Concentration Assay, Isolation, Diagnostic Assay

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

    Techniques Used: Plasmid Preparation, Incubation

    30) Product Images from "Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice"

    Article Title: Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20041074

    DC-PCR analysis of genomic switch recombination in wild type and Atm −/− B cells. Genomic DNA was isolated from B cells activated in vitro for 6 d, digested with EcoRI, and ligated with T4 DNA ligase. Twofold serial dilutions were used as a template for DC-PCR using primers specific for the recombined S regions. nAChR levels were also determined by DC-PCR to control for equal template loading. The results shown are representative of two independent experiments.
    Figure Legend Snippet: DC-PCR analysis of genomic switch recombination in wild type and Atm −/− B cells. Genomic DNA was isolated from B cells activated in vitro for 6 d, digested with EcoRI, and ligated with T4 DNA ligase. Twofold serial dilutions were used as a template for DC-PCR using primers specific for the recombined S regions. nAChR levels were also determined by DC-PCR to control for equal template loading. The results shown are representative of two independent experiments.

    Techniques Used: Polymerase Chain Reaction, Isolation, In Vitro

    31) Product Images from "Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines"

    Article Title: Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki154

    Effect of CPT treatment on DSB repair efficiency in CRC cell lines. Cells were treated with 100 nM CPT just after transfection and until plasmid recovery. Histograms represent the mean percentage of repair efficiency established from a minimum of three independent experiments for plasmids linearized with ApaI ( A ), EcoRI ( B ) and ApaI-EcoRI ( C ). The numbers of independent experiments are marked above each histogram for each CRC cell line tested. Only P values statistically different are specified.
    Figure Legend Snippet: Effect of CPT treatment on DSB repair efficiency in CRC cell lines. Cells were treated with 100 nM CPT just after transfection and until plasmid recovery. Histograms represent the mean percentage of repair efficiency established from a minimum of three independent experiments for plasmids linearized with ApaI ( A ), EcoRI ( B ) and ApaI-EcoRI ( C ). The numbers of independent experiments are marked above each histogram for each CRC cell line tested. Only P values statistically different are specified.

    Techniques Used: Cycling Probe Technology, Transfection, Plasmid Preparation

    End-joining efficiency of DSB repair by NHEJ in MMR-deficient and MMR-proficient CRC cell lines. Histograms represent the mean percentage of repair efficiency established from a minimum of three independent experiments using plasmids linearized with ApaI ( A ), EcoRI ( B ) and ApaI-EcoRI ( C ). The number of independent experiments is shown above each histogram. The P values refer to the comparison of all cell lines.
    Figure Legend Snippet: End-joining efficiency of DSB repair by NHEJ in MMR-deficient and MMR-proficient CRC cell lines. Histograms represent the mean percentage of repair efficiency established from a minimum of three independent experiments using plasmids linearized with ApaI ( A ), EcoRI ( B ) and ApaI-EcoRI ( C ). The number of independent experiments is shown above each histogram. The P values refer to the comparison of all cell lines.

    Techniques Used: Non-Homologous End Joining

    End-joining fidelity of DSB repair by NHEJ in MMR-deficient and MMR-proficient CRC cell lines. Histograms represent the mean frequency in percentage of the error-free events after repair of DSB induced by ApaI ( A ) or EcoRI ( B ) established from a minimum of three independent experiments and a minimum of 250 bacterial colonies analyzed. The numbers of bacterial colonies analyzed are specified above the histograms for each CRC cell lines tested. The P values refer to the comparison of all cell lines.
    Figure Legend Snippet: End-joining fidelity of DSB repair by NHEJ in MMR-deficient and MMR-proficient CRC cell lines. Histograms represent the mean frequency in percentage of the error-free events after repair of DSB induced by ApaI ( A ) or EcoRI ( B ) established from a minimum of three independent experiments and a minimum of 250 bacterial colonies analyzed. The numbers of bacterial colonies analyzed are specified above the histograms for each CRC cell lines tested. The P values refer to the comparison of all cell lines.

    Techniques Used: Non-Homologous End Joining

    32) Product Images from "Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿"

    Article Title: Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02797-06

    Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.
    Figure Legend Snippet: Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.

    Techniques Used: Southern Blot, Molecular Weight, Infection, Methylation, CpG Methylation Assay

    33) Product Images from "Role of the Agr-Like Quorum-Sensing System in Regulating Toxin Production by Clostridium perfringens Type B Strains CN1793 and CN1795"

    Article Title: Role of the Agr-Like Quorum-Sensing System in Regulating Toxin Production by Clostridium perfringens Type B Strains CN1793 and CN1795

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00438-12

    Construction of agrB null mutants in C. perfringens type B strains CN1793 and CN1795 by intron-based insertional mutagenesis. PCR analyses using primers to internal agrB ORF sequences and DNA from wild-type CN1795, the agrB null mutant (CN1795:: agrB ), or the complemented strain CN1795:: agrB (p3) (A) or from wild-type CN1793, the agrB null mutant (CN1793:: agrB ), or the complemented strain CN1793:: agrB (p3) (B). (C) Southern blot hybridization analysis for the presence of a group II intron insertion in wild-type CN1793 or CN1795, the agrB null mutants of those strains, and complementing strains. DNA from each strain was digested with EcoRI, electrophoresed on a 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with a DIG-labeled intron-specific probe. Sizes of DNA fragments in kilobases (kb) are shown to the left. (D) RT-PCR analyses for agrB mRNA expression by wild-type CN1795, CN1795:: agrB , or CN1795:: agrB (p3). Sample RNA was collected from 4-h TGY cultures. As indicated, reverse transcriptase (RT) was (+) or was not (−) added to the reaction tubes. DNA polymerase III ( polC ) served as an internal control. (E) Similar RT-PCR analyses for agrB mRNA expression by wild-type CN1793, CN1793:: agrB , or CN1793:: agrB (p3).
    Figure Legend Snippet: Construction of agrB null mutants in C. perfringens type B strains CN1793 and CN1795 by intron-based insertional mutagenesis. PCR analyses using primers to internal agrB ORF sequences and DNA from wild-type CN1795, the agrB null mutant (CN1795:: agrB ), or the complemented strain CN1795:: agrB (p3) (A) or from wild-type CN1793, the agrB null mutant (CN1793:: agrB ), or the complemented strain CN1793:: agrB (p3) (B). (C) Southern blot hybridization analysis for the presence of a group II intron insertion in wild-type CN1793 or CN1795, the agrB null mutants of those strains, and complementing strains. DNA from each strain was digested with EcoRI, electrophoresed on a 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with a DIG-labeled intron-specific probe. Sizes of DNA fragments in kilobases (kb) are shown to the left. (D) RT-PCR analyses for agrB mRNA expression by wild-type CN1795, CN1795:: agrB , or CN1795:: agrB (p3). Sample RNA was collected from 4-h TGY cultures. As indicated, reverse transcriptase (RT) was (+) or was not (−) added to the reaction tubes. DNA polymerase III ( polC ) served as an internal control. (E) Similar RT-PCR analyses for agrB mRNA expression by wild-type CN1793, CN1793:: agrB , or CN1793:: agrB (p3).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing

    Related Articles

    Ligation:

    Article Title: High-throughput screening of soluble recombinant proteins
    Article Snippet: A specific cleavage sequence of protease (e.g., thrombin or factor Xa) was introduced immediately after the EcoRI site and before the coding sequence of target protein; this was achieved by stringent design of the sticky-end PCR primers. .. 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). .. Plasmid DNA purification was performed in a 96-well format using Millpore's Motage plasmid miniprep kit.

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

    Isolation:

    Article Title: Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere
    Article Snippet: Nitrate-regulated ice nucleation activity of DH5α, 299R, and EcCT501R containing pNice was assessed in M9 with various concentrations of sodium nitrate by using methods mentioned above. .. The inaZ gene was removed from E. cloacae (pNice) by digestion with EcoRI and NotI (New England Biolabs) and replaced with a GFP reporter gene isolated as an EcoRI and NotI restriction digest fragment from pPROBE-NT ( ) to produce pNgfp. .. This plasmid was transformed into E. coli strain DH5α by electroporation under standard conditions ( ).

    Modification:

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

    Plasmid Preparation:

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

    Amplification:

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: The first selective amplification consisted of 12 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 60 s, in which the annealing temperature was lowered by 0.7 °C per cycle, followed by 23 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 60 s. The cDNA-AFLP products were electrophoresed in 8% nondenaturing polyacrylamide gels at 240 V for 2.5 h in 1× TBE buffer and then visualized via silver staining. .. Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. ..

    Incubation:

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: The first selective amplification consisted of 12 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 60 s, in which the annealing temperature was lowered by 0.7 °C per cycle, followed by 23 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 60 s. The cDNA-AFLP products were electrophoresed in 8% nondenaturing polyacrylamide gels at 240 V for 2.5 h in 1× TBE buffer and then visualized via silver staining. .. Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. ..

    Subcloning:

    Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells
    Article Snippet: .. Sub-Cloning of Genes to pMSCV To perform sub-cloning, Hras V12 and SV40 large T antigene (LTg) were separated from pBABE-Hras V12 and pBABE-SV40 LTg by the enzymatic digestion with BamHI and EcoRI (NEB, Ipswich, MA), or with BamHI (NEB), respectively. .. Integration of Hras V12 and SV40LTg into either pMSCV-GFP or pMSCV-RFP was performed by ligation with T4-ligase (NEB) and produced pMSCV-Hras V12-GFP and pMSCV-SV40 LTg-RFP.

    Purification:

    Article Title: Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101
    Article Snippet: The PCR for preparing the complementing strain used LongAmp Taq polymerase (New England Biolabs) and the following parameters: (i) 95°C for 4 min; (ii) 35 cycles of 95°C for 30 s, 50°C for 30 s, and 65°C for 2 min; and (iii) a final extension of 65°C for 10 min. PCRs for screening mutants/transformants were performed using 2× MasterMix Taq polymerase (New England Biolabs) and the following parameters: (i) 94°C for 4 min; (ii) 35 cycles of 94°C for 30 s, 50°C for 30 s, and 68°C for 2 min; and (iii) a final extension of 68°C for 10 min. To confirm the insertion of only a single intron into SM101-CPR0195KO or SM101-CPR1055KO, Southern blot analysis was performed as previously described ( ). .. 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. .. An intron-specific DIG-labeled probe was then incubated with the blot, and the hybridized probe was detected with CPSD substrate (Roche).

    Agarose Gel Electrophoresis:

    Article Title: Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101
    Article Snippet: The PCR for preparing the complementing strain used LongAmp Taq polymerase (New England Biolabs) and the following parameters: (i) 95°C for 4 min; (ii) 35 cycles of 95°C for 30 s, 50°C for 30 s, and 65°C for 2 min; and (iii) a final extension of 65°C for 10 min. PCRs for screening mutants/transformants were performed using 2× MasterMix Taq polymerase (New England Biolabs) and the following parameters: (i) 94°C for 4 min; (ii) 35 cycles of 94°C for 30 s, 50°C for 30 s, and 68°C for 2 min; and (iii) a final extension of 68°C for 10 min. To confirm the insertion of only a single intron into SM101-CPR0195KO or SM101-CPR1055KO, Southern blot analysis was performed as previously described ( ). .. 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. .. An intron-specific DIG-labeled probe was then incubated with the blot, and the hybridized probe was detected with CPSD substrate (Roche).

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    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 <t>DNA</t> 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 <t>EcoRI</t> 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.
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    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

    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 .

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0119221

    Figure Lengend 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 .

    Article Snippet: Restriction endonucleases PvuII, EcoRI and HindIII were purchased from New England Biolabs (Ipswich, MA, USA) and used according to manufacturer’s instructions.

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

    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

    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