colony pcrs Search Results


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  • 99
    Qiagen colony pcr
    Colony Pcr, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher high resolution melting hrm colony pcrs
    High Resolution Melting Hrm Colony Pcrs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Eton Bioscience colony pcr
    Colony Pcr, supplied by Eton Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher colony pcr
    Colony Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Epicentre Biotechnologies colony pcr
    Colony Pcr, supplied by Epicentre Biotechnologies, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Genewiz colony pcr amplification
    Colony Pcr Amplification, supplied by Genewiz, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa colony pcr analysis
    Colony Pcr Analysis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Eurofins colony pcr
    Colony Pcr, supplied by Eurofins, used in various techniques. Bioz Stars score: 99/100, based on 238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GATC Biotech colony pcr
    Pilin variation in antigenic variation deficient strains. Cells were picked from ( a ) inoculation zone, and ( b ) outgrowth, respectively. After dilution and growth on agar plates, individual colonies were picked and <t>pilE</t> was sequenced. Sequences were categorised and the fraction shown is the number of sequences per category normalised by the total number of sequences found for each strain. Full length: most abundant sequence in inoculum; full length, av: sequence change mappable to pilS sequence; truncated, pv: length change in poly-C sequence causing premature stop-codon; no <t>PCR:</t> PCR amplification did not result in a product. Grey: red wt (Ng106) and green wt (Ng165), green: green recA (Ng167) and red recA (Ng168), red: green ∆G4 (Ng169) and red ΔG4 (Ng170). Mean and standard error of three independent experiments.
    Colony Pcr, supplied by GATC Biotech, used in various techniques. Bioz Stars score: 94/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Genewiz colony pcr
    TALEN design and Gene Editing using <t>TALENs</t> and ssODNs. (A). The TALEN pair, designed and built using the Golden Gate method, induces a double stranded break immediately preceding the mutant codon. RVDs are shown as color coded binding blocks next to their respective base, yellow NI:A, green NG:T, blue HD:C and red NN:G. Fok1 domains are shown in black and are positioned at their predicted cut site. (B). Unsynchronized HCT116-19 cells were harvested electroporated at a concentration of 5e5 cells/100 ul with TALENs and/or 72NT ODN at the indicated amounts. TALEN amounts reflect the total TALEN plasmid added to each sample in equal portions (1 ug L848-19 and 1 ug R898-19). Left and Right TALENs must be present for a DSB to be made. Following electroporation, cells were placed in 6-well plates and allowed to recover for 48 hours. Analyses took place on a Guava EasyCyte 5 HT flow cytometer (see Materials and Methods). Correction efficiency (%) was determined by the number of viable eGFP positive cells divided by the total viable cells in the population. Each treatment was performed in triplicate and error bars represent standard error. (C). Synchronized HCT116-19 cells were electroporated under the following conditions; 2 ug TALEN and 1.35 ug 72NT at 5e5 cells/100 ul. Cells were then sorted for GFP+ at 24, 48, 72 and 240 hours post electroporation. Immediately following cell sorting, DNA was isolated and the region surrounding the target base was amplified via <t>PCR.</t> Samp les were submitted to Genewiz (South Plainfield, NJ) for sequencing analysis.
    Colony Pcr, supplied by Genewiz, used in various techniques. Bioz Stars score: 94/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Macrogen colony pcr
    Correlation Between Real-Time <t>PCR</t> and ELISA Results in <t>igVR-1a</t>
    Colony Pcr, supplied by Macrogen, used in various techniques. Bioz Stars score: 96/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    MWG-Biotech colony pcr
    Correlation Between Real-Time <t>PCR</t> and ELISA Results in <t>igVR-1a</t>
    Colony Pcr, supplied by MWG-Biotech, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Toyobo colony pcr
    EMSA analysis of <t>Sco5333</t> to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is <t>PCR</t> amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.
    Colony Pcr, supplied by Toyobo, used in various techniques. Bioz Stars score: 98/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AGOWA GmbH colony pcr
    EMSA analysis of <t>Sco5333</t> to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is <t>PCR</t> amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.
    Colony Pcr, supplied by AGOWA GmbH, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Elim Bio colony pcr
    EMSA analysis of <t>Sco5333</t> to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is <t>PCR</t> amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.
    Colony Pcr, supplied by Elim Bio, used in various techniques. Bioz Stars score: 87/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Microsynth colony pcr
    EMSA analysis of <t>Sco5333</t> to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is <t>PCR</t> amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.
    Colony Pcr, supplied by Microsynth, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore colony pcr
    EMSA analysis of <t>Sco5333</t> to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is <t>PCR</t> amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.
    Colony Pcr, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega colony pcr
    Generation of gene deletions in E. faecalis . (a) Genetic environment of a heterodimeric ABC transporter gene cluster ( efrCD is presented as an example) in the context of upstream ( ef0786 , ef0787 ) and downstream ( ef0791 , ef0792 ) genes. Upstream and downstream flanking regions (∼1,000 bp each) were amplified from genomic <t>DNA</t> (gDNA) and were cloned into the gene deletion vector pCJK245_FX. KO, knockout. (b) Translational products of gene remnants after transporter gene deletion. (c) Confirmation of transporter gene deletions in E. faecalis by <t>PCR</t> amplification from wild-type (∼6,000 bp) and mutant (∼2,500 bp) genomic DNA with primers used to amplify the upstream and downstream regions (5′-FW and 3′-RV [see Table S2 in the supplemental material]). Gene deletions for all seven heterodimeric ABC exporters were generated in E. faecalis 4205. The two transporter genes efrAB and efrCD were also deleted in E. faecalis V583.
    Colony Pcr, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 484 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sangon Biotech colony pcr
    Generation of gene deletions in E. faecalis . (a) Genetic environment of a heterodimeric ABC transporter gene cluster ( efrCD is presented as an example) in the context of upstream ( ef0786 , ef0787 ) and downstream ( ef0791 , ef0792 ) genes. Upstream and downstream flanking regions (∼1,000 bp each) were amplified from genomic <t>DNA</t> (gDNA) and were cloned into the gene deletion vector pCJK245_FX. KO, knockout. (b) Translational products of gene remnants after transporter gene deletion. (c) Confirmation of transporter gene deletions in E. faecalis by <t>PCR</t> amplification from wild-type (∼6,000 bp) and mutant (∼2,500 bp) genomic DNA with primers used to amplify the upstream and downstream regions (5′-FW and 3′-RV [see Table S2 in the supplemental material]). Gene deletions for all seven heterodimeric ABC exporters were generated in E. faecalis 4205. The two transporter genes efrAB and efrCD were also deleted in E. faecalis V583.
    Colony Pcr, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 97/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Genaxxon BioScience GmbH colony pcr
    Generation of gene deletions in E. faecalis . (a) Genetic environment of a heterodimeric ABC transporter gene cluster ( efrCD is presented as an example) in the context of upstream ( ef0786 , ef0787 ) and downstream ( ef0791 , ef0792 ) genes. Upstream and downstream flanking regions (∼1,000 bp each) were amplified from genomic <t>DNA</t> (gDNA) and were cloned into the gene deletion vector pCJK245_FX. KO, knockout. (b) Translational products of gene remnants after transporter gene deletion. (c) Confirmation of transporter gene deletions in E. faecalis by <t>PCR</t> amplification from wild-type (∼6,000 bp) and mutant (∼2,500 bp) genomic DNA with primers used to amplify the upstream and downstream regions (5′-FW and 3′-RV [see Table S2 in the supplemental material]). Gene deletions for all seven heterodimeric ABC exporters were generated in E. faecalis 4205. The two transporter genes efrAB and efrCD were also deleted in E. faecalis V583.
    Colony Pcr, supplied by Genaxxon BioScience GmbH, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Laragen Inc colony pcr
    Generation of gene deletions in E. faecalis . (a) Genetic environment of a heterodimeric ABC transporter gene cluster ( efrCD is presented as an example) in the context of upstream ( ef0786 , ef0787 ) and downstream ( ef0791 , ef0792 ) genes. Upstream and downstream flanking regions (∼1,000 bp each) were amplified from genomic <t>DNA</t> (gDNA) and were cloned into the gene deletion vector pCJK245_FX. KO, knockout. (b) Translational products of gene remnants after transporter gene deletion. (c) Confirmation of transporter gene deletions in E. faecalis by <t>PCR</t> amplification from wild-type (∼6,000 bp) and mutant (∼2,500 bp) genomic DNA with primers used to amplify the upstream and downstream regions (5′-FW and 3′-RV [see Table S2 in the supplemental material]). Gene deletions for all seven heterodimeric ABC exporters were generated in E. faecalis 4205. The two transporter genes efrAB and efrCD were also deleted in E. faecalis V583.
    Colony Pcr, supplied by Laragen Inc, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pilin variation in antigenic variation deficient strains. Cells were picked from ( a ) inoculation zone, and ( b ) outgrowth, respectively. After dilution and growth on agar plates, individual colonies were picked and pilE was sequenced. Sequences were categorised and the fraction shown is the number of sequences per category normalised by the total number of sequences found for each strain. Full length: most abundant sequence in inoculum; full length, av: sequence change mappable to pilS sequence; truncated, pv: length change in poly-C sequence causing premature stop-codon; no PCR: PCR amplification did not result in a product. Grey: red wt (Ng106) and green wt (Ng165), green: green recA (Ng167) and red recA (Ng168), red: green ∆G4 (Ng169) and red ΔG4 (Ng170). Mean and standard error of three independent experiments.

    Journal: Scientific Reports

    Article Title: Phase and antigenic variation govern competition dynamics through positioning in bacterial colonies

    doi: 10.1038/s41598-017-12472-7

    Figure Lengend Snippet: Pilin variation in antigenic variation deficient strains. Cells were picked from ( a ) inoculation zone, and ( b ) outgrowth, respectively. After dilution and growth on agar plates, individual colonies were picked and pilE was sequenced. Sequences were categorised and the fraction shown is the number of sequences per category normalised by the total number of sequences found for each strain. Full length: most abundant sequence in inoculum; full length, av: sequence change mappable to pilS sequence; truncated, pv: length change in poly-C sequence causing premature stop-codon; no PCR: PCR amplification did not result in a product. Grey: red wt (Ng106) and green wt (Ng165), green: green recA (Ng167) and red recA (Ng168), red: green ∆G4 (Ng169) and red ΔG4 (Ng170). Mean and standard error of three independent experiments.

    Article Snippet: The pilE fragment amplified by colony PCR was sequenced by a DNA sequencing service (GATC Biotech AG., Konstanz, Germany).

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification

    Pilin antigenic and phase variation. red wt (Ng106) and green wt (Ng165) were picked from the initial inoculum, the inoculation zone (48 h), and the outgrowth (48 h), respectively. After dilution and growth on agar plates, individual colonies (clones) were picked and pilE was sequenced. Sequences were categorised and the fraction shown is the number of sequences per category normalised by the total number of sequences of inoculum, inoculation zone and outgrowth, respectively. Full length: most abundant sequence in inoculum; full length, av: sequence change mappable to pilS sequence; truncated, pv: length change in poly-C sequence causing premature stop-codon; no PCR: PCR amplification did not result in a product. Mean and standard error of three independent experiments.

    Journal: Scientific Reports

    Article Title: Phase and antigenic variation govern competition dynamics through positioning in bacterial colonies

    doi: 10.1038/s41598-017-12472-7

    Figure Lengend Snippet: Pilin antigenic and phase variation. red wt (Ng106) and green wt (Ng165) were picked from the initial inoculum, the inoculation zone (48 h), and the outgrowth (48 h), respectively. After dilution and growth on agar plates, individual colonies (clones) were picked and pilE was sequenced. Sequences were categorised and the fraction shown is the number of sequences per category normalised by the total number of sequences of inoculum, inoculation zone and outgrowth, respectively. Full length: most abundant sequence in inoculum; full length, av: sequence change mappable to pilS sequence; truncated, pv: length change in poly-C sequence causing premature stop-codon; no PCR: PCR amplification did not result in a product. Mean and standard error of three independent experiments.

    Article Snippet: The pilE fragment amplified by colony PCR was sequenced by a DNA sequencing service (GATC Biotech AG., Konstanz, Germany).

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification

    TALEN design and Gene Editing using TALENs and ssODNs. (A). The TALEN pair, designed and built using the Golden Gate method, induces a double stranded break immediately preceding the mutant codon. RVDs are shown as color coded binding blocks next to their respective base, yellow NI:A, green NG:T, blue HD:C and red NN:G. Fok1 domains are shown in black and are positioned at their predicted cut site. (B). Unsynchronized HCT116-19 cells were harvested electroporated at a concentration of 5e5 cells/100 ul with TALENs and/or 72NT ODN at the indicated amounts. TALEN amounts reflect the total TALEN plasmid added to each sample in equal portions (1 ug L848-19 and 1 ug R898-19). Left and Right TALENs must be present for a DSB to be made. Following electroporation, cells were placed in 6-well plates and allowed to recover for 48 hours. Analyses took place on a Guava EasyCyte 5 HT flow cytometer (see Materials and Methods). Correction efficiency (%) was determined by the number of viable eGFP positive cells divided by the total viable cells in the population. Each treatment was performed in triplicate and error bars represent standard error. (C). Synchronized HCT116-19 cells were electroporated under the following conditions; 2 ug TALEN and 1.35 ug 72NT at 5e5 cells/100 ul. Cells were then sorted for GFP+ at 24, 48, 72 and 240 hours post electroporation. Immediately following cell sorting, DNA was isolated and the region surrounding the target base was amplified via PCR. Samp les were submitted to Genewiz (South Plainfield, NJ) for sequencing analysis.

    Journal: Scientific Reports

    Article Title: Combinatorial gene editing in mammalian cells using ssODNs and TALENs

    doi: 10.1038/srep03791

    Figure Lengend Snippet: TALEN design and Gene Editing using TALENs and ssODNs. (A). The TALEN pair, designed and built using the Golden Gate method, induces a double stranded break immediately preceding the mutant codon. RVDs are shown as color coded binding blocks next to their respective base, yellow NI:A, green NG:T, blue HD:C and red NN:G. Fok1 domains are shown in black and are positioned at their predicted cut site. (B). Unsynchronized HCT116-19 cells were harvested electroporated at a concentration of 5e5 cells/100 ul with TALENs and/or 72NT ODN at the indicated amounts. TALEN amounts reflect the total TALEN plasmid added to each sample in equal portions (1 ug L848-19 and 1 ug R898-19). Left and Right TALENs must be present for a DSB to be made. Following electroporation, cells were placed in 6-well plates and allowed to recover for 48 hours. Analyses took place on a Guava EasyCyte 5 HT flow cytometer (see Materials and Methods). Correction efficiency (%) was determined by the number of viable eGFP positive cells divided by the total viable cells in the population. Each treatment was performed in triplicate and error bars represent standard error. (C). Synchronized HCT116-19 cells were electroporated under the following conditions; 2 ug TALEN and 1.35 ug 72NT at 5e5 cells/100 ul. Cells were then sorted for GFP+ at 24, 48, 72 and 240 hours post electroporation. Immediately following cell sorting, DNA was isolated and the region surrounding the target base was amplified via PCR. Samp les were submitted to Genewiz (South Plainfield, NJ) for sequencing analysis.

    Article Snippet: Antibiotic selection and colony PCR was performed to confirm correctly constructed TALENs, and corrected clones were sent for sequence confirmation (Genewiz Inc, South Plainfield, NJ). represents a schematic of the TALEN used in these studies.

    Techniques: TALENs, Mutagenesis, Binding Assay, Concentration Assay, Plasmid Preparation, Electroporation, Flow Cytometry, Cytometry, FACS, Isolation, Amplification, Polymerase Chain Reaction, Sequencing

    Correlation Between Real-Time PCR and ELISA Results in igVR-1a

    Journal: Hepatitis Monthly

    Article Title: Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region

    doi: 10.5812/hepatmon.38261

    Figure Lengend Snippet: Correlation Between Real-Time PCR and ELISA Results in igVR-1a

    Article Snippet: All plasmids of the igVR-1a chimera were then confirmed by colony PCR with forward and reverse specific primers, as well as sequencing (Macrogen, Korea).

    Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    EMSA analysis of Sco5333 to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is PCR amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.

    Journal: Nucleic Acids Research

    Article Title: Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

    doi: 10.1093/nar/gku1376

    Figure Lengend Snippet: EMSA analysis of Sco5333 to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is PCR amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.

    Article Snippet: Over-expression and purification of Sco5333, Tbis1 and Dcm methyltransferase Dcm coding sequence was amplified by colony-PCR from E. coli DH10B using KOD DNA polymerase (TOYOBO) and primers dcmEX-F & R, the PCR fragment was treated with NdeI and EcoRI and ligated into the expression vector pET15b, generating pJTU4357 for producing N-terminally His6-tagged Dcm.

    Techniques: Methylation, Derivative Assay, Sequencing, Polymerase Chain Reaction, Amplification, Modification, Gel Permeation Chromatography, Synthesized, Titration, Concentration Assay, Binding Assay

    Generation of gene deletions in E. faecalis . (a) Genetic environment of a heterodimeric ABC transporter gene cluster ( efrCD is presented as an example) in the context of upstream ( ef0786 , ef0787 ) and downstream ( ef0791 , ef0792 ) genes. Upstream and downstream flanking regions (∼1,000 bp each) were amplified from genomic DNA (gDNA) and were cloned into the gene deletion vector pCJK245_FX. KO, knockout. (b) Translational products of gene remnants after transporter gene deletion. (c) Confirmation of transporter gene deletions in E. faecalis by PCR amplification from wild-type (∼6,000 bp) and mutant (∼2,500 bp) genomic DNA with primers used to amplify the upstream and downstream regions (5′-FW and 3′-RV [see Table S2 in the supplemental material]). Gene deletions for all seven heterodimeric ABC exporters were generated in E. faecalis 4205. The two transporter genes efrAB and efrCD were also deleted in E. faecalis V583.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

    doi: 10.1128/AAC.00661-16

    Figure Lengend Snippet: Generation of gene deletions in E. faecalis . (a) Genetic environment of a heterodimeric ABC transporter gene cluster ( efrCD is presented as an example) in the context of upstream ( ef0786 , ef0787 ) and downstream ( ef0791 , ef0792 ) genes. Upstream and downstream flanking regions (∼1,000 bp each) were amplified from genomic DNA (gDNA) and were cloned into the gene deletion vector pCJK245_FX. KO, knockout. (b) Translational products of gene remnants after transporter gene deletion. (c) Confirmation of transporter gene deletions in E. faecalis by PCR amplification from wild-type (∼6,000 bp) and mutant (∼2,500 bp) genomic DNA with primers used to amplify the upstream and downstream regions (5′-FW and 3′-RV [see Table S2 in the supplemental material]). Gene deletions for all seven heterodimeric ABC exporters were generated in E. faecalis 4205. The two transporter genes efrAB and efrCD were also deleted in E. faecalis V583.

    Article Snippet: White colonies (in total 600 for the seven deletion mutants) were screened for gene deletions using colony PCR (with GoTaq G2 DNA polymerase; Promega).

    Techniques: Amplification, Clone Assay, Plasmid Preparation, Knock-Out, Polymerase Chain Reaction, Mutagenesis, Generated

    User interfaces of the cloning database

    Journal: BMC Genomics

    Article Title: The full-ORF clone resource of the German cDNA Consortium

    doi: 10.1186/1471-2164-8-399

    Figure Lengend Snippet: User interfaces of the cloning database "SCISSORS" . A: Screenshot of the data entry sheet of second step ORF PCR. B: 96-well colony PCR plate assembled by the software. The entered PCR results are automatically color-coded by the software as follows: red and grey: positive or negative colony (presence or absence of a band of expected size on the agarose gel), blue: entry clone colonies already used for plasmid preparation, yellow: colonies selected for generation of a new entry clone 96-well plate. C: User interface of entry clone plates. Clones scored positive in the control digest are automatically color-coded in green, negative clones remain white. Clicking on the plate positions opens a window to enter the sequencing result of the particular entry clone. D: Results of a working step can also be entered in a table format, as shown for the entry clone validation.

    Article Snippet: Eight colonies per ORF were analysed for the presence of the ORF of expected size in a colony PCR, utilizing the Perkin Elmer Multiprobe robot to set-up the reactions.

    Techniques: Clone Assay, Polymerase Chain Reaction, Software, Agarose Gel Electrophoresis, Plasmid Preparation, Sequencing

    PCR-success with and without optimization of the reaction conditions . The impact of the optimizations steps on the success rates (in percentages) are shown in dependence on the ORF size. A PCR was defined successful when a DNA product of the expected size was observed in analytical agarose gel electrophoresis.

    Journal: BMC Genomics

    Article Title: The full-ORF clone resource of the German cDNA Consortium

    doi: 10.1186/1471-2164-8-399

    Figure Lengend Snippet: PCR-success with and without optimization of the reaction conditions . The impact of the optimizations steps on the success rates (in percentages) are shown in dependence on the ORF size. A PCR was defined successful when a DNA product of the expected size was observed in analytical agarose gel electrophoresis.

    Article Snippet: Eight colonies per ORF were analysed for the presence of the ORF of expected size in a colony PCR, utilizing the Perkin Elmer Multiprobe robot to set-up the reactions.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Comparison of PCR products amplified with two different DNA polymerase systems . A total of 100 ORFs (50 ORFs per enzyme mix), ranging from 300 to 4,000 bp in size, were amplified. Electrophoretic analysis of 10 representative ORFs amplified using either the supplier I (left panel) or the Phusion™ High-Fidelity DNA Polymerase (Finnzymes) (right panel). One-tenth of each reaction product of first and second step ORF amplification were loaded adjacent to each other on an analytical agarose gel. According to the lane number the expected ORF sizes and accession numbers of first-step PCR templates are as follows: 1: 759 bp, BC100921; 2: 1125 bp, BC093648; 3: 1554 bp, BC104948; 4: 3198 bp, BC117368; 5: 651 bp, BC105131; 6: 1653 bp, BC109061; 7: 2400 bp, BC113416; 8: 1737 bp, BC117320; 9: 1854 bp, BC101755; 10: 720 bp, BC113739.'M' indicates the molecular weight marker lanes.

    Journal: BMC Genomics

    Article Title: The full-ORF clone resource of the German cDNA Consortium

    doi: 10.1186/1471-2164-8-399

    Figure Lengend Snippet: Comparison of PCR products amplified with two different DNA polymerase systems . A total of 100 ORFs (50 ORFs per enzyme mix), ranging from 300 to 4,000 bp in size, were amplified. Electrophoretic analysis of 10 representative ORFs amplified using either the supplier I (left panel) or the Phusion™ High-Fidelity DNA Polymerase (Finnzymes) (right panel). One-tenth of each reaction product of first and second step ORF amplification were loaded adjacent to each other on an analytical agarose gel. According to the lane number the expected ORF sizes and accession numbers of first-step PCR templates are as follows: 1: 759 bp, BC100921; 2: 1125 bp, BC093648; 3: 1554 bp, BC104948; 4: 3198 bp, BC117368; 5: 651 bp, BC105131; 6: 1653 bp, BC109061; 7: 2400 bp, BC113416; 8: 1737 bp, BC117320; 9: 1854 bp, BC101755; 10: 720 bp, BC113739.'M' indicates the molecular weight marker lanes.

    Article Snippet: Eight colonies per ORF were analysed for the presence of the ORF of expected size in a colony PCR, utilizing the Perkin Elmer Multiprobe robot to set-up the reactions.

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Cloning strategy for the simultaneous generation of entry clones in open and closed configuration . A : Sequences of entry clones 3' of the ORF either containing or not containing a stop codon. The sequences correspond to the reverse primer sequences of 2-step PCR. In presence of an A at the degenerated position, a stop codon is created and the Bam HI site (underlined) destroyed. In contrast, the inclusion of a G generates a Bam HI site and results in a translational read-through. B : Schematic presentation of the entry clone map. 'for' and 'rev' indicate the binding sites of the colony PCR primers. The degenerated position is indicated by the arrow. C : Bam HI colony-PCR restriction digest of eight independent colonies resulting from BP cloning of four different ORFs amplified using degenerated reverse primers. The arrows mark the additional band which appears in presence of the Bam HI recognition sequence, indicating that the ORF does not contain a stop codon. ORF 4 contains an internal BamHI site indicated by the appearance of a band of about 100 bp. 'M' indicates the molecular weight marker lanes.

    Journal: BMC Genomics

    Article Title: The full-ORF clone resource of the German cDNA Consortium

    doi: 10.1186/1471-2164-8-399

    Figure Lengend Snippet: Cloning strategy for the simultaneous generation of entry clones in open and closed configuration . A : Sequences of entry clones 3' of the ORF either containing or not containing a stop codon. The sequences correspond to the reverse primer sequences of 2-step PCR. In presence of an A at the degenerated position, a stop codon is created and the Bam HI site (underlined) destroyed. In contrast, the inclusion of a G generates a Bam HI site and results in a translational read-through. B : Schematic presentation of the entry clone map. 'for' and 'rev' indicate the binding sites of the colony PCR primers. The degenerated position is indicated by the arrow. C : Bam HI colony-PCR restriction digest of eight independent colonies resulting from BP cloning of four different ORFs amplified using degenerated reverse primers. The arrows mark the additional band which appears in presence of the Bam HI recognition sequence, indicating that the ORF does not contain a stop codon. ORF 4 contains an internal BamHI site indicated by the appearance of a band of about 100 bp. 'M' indicates the molecular weight marker lanes.

    Article Snippet: Eight colonies per ORF were analysed for the presence of the ORF of expected size in a colony PCR, utilizing the Perkin Elmer Multiprobe robot to set-up the reactions.

    Techniques: Clone Assay, Polymerase Chain Reaction, Binding Assay, Amplification, Sequencing, Molecular Weight, Marker